Fc-region variants with modified FcRn-binding and methods of use

ABSTRACT

Herein is reported an IgG class Fc-region comprising a first variant Fc-region polypeptide and a second variant Fc-region polypeptide, wherein a) the first variant Fc-region polypeptide is derived from a first parent IgG class Fc-region polypeptide and the second variant Fc-region polypeptide is derived from a second parent IgG class Fc-region polypeptide, whereby the first parent IgG class Fc-region polypeptide is identical to or different from the second parent IgG class Fc-region polypeptide, and b) the first variant Fc-region polypeptide differs from the second variant Fc-region polypeptide in one or more amino acid residues other than those amino acid residues in which the first parent IgG class Fc-region polypeptide differs from the second parent IgG class Fc-region polypeptide, and c) the IgG class Fc-region comprising the first variant Fc-region polypeptide and the second variant Fc-region polypeptide has an affinity to a human Fc-receptor that is different than that of an IgG class Fc-region comprising the first parent IgG class Fc-region polypeptide of a) and the second parent IgG class Fc-region polypeptide of a), wherein either the first Fc-region polypeptide or the second Fc-region polypeptide or both Fc-region polypeptides comprise independently of each other one of the following mutations or combination of mutations: T307H, or Q311H, or E430H, or N434H, or T307H and Q311H, or T307H and E430H, or T307H and N434A, or T307H and N434H, or T307Q and Q311H, or T307Q and E430H, or T307Q and N434H, or T307H and Q311H and E430H and N434A, or T307H and Q311H and E430H and N434H, or T307H and Q311H and E430H and N434Y, or T307Q and Q311H and E430H and N434A, or T307Q and Q311H and E430H and N434H, or T307Q and Q311H and E430H and N434Y, or T307Q and V308P and N434Y and Y436H, or T307H and M252Y and S254T and T256E, or T307Q and M252Y and S254T and T256E, or Q311H and M252Y and S254T and T256E, or E430 H and M252Y and S254T and T256E, or N434H and M252Y and S254T and T256E, or T307H and Q311H and M252Y and S254T and T256E, or T307H and E430H and M252Y and S254T and T256E, or T307H and N434A and M252Y and S254T and T256E, or T307H and N434H and M252Y and S254T and T256E, or T307Q and Q311H and M252Y and S254T and T256E, or T307Q and E430H and M252Y and S254T and T256E, or T307Q and N434H and M252Y and S254T and T256E, or T307H and Q311H and E430H and N434A and M252Y and S254T and T256E, or T307H and Q311H and E430H and N434H and M252Y and S254T and T256E, or T307H and Q311H and E430H and N434Y and M252Y and S254T and T256E, or T307Q and Q311H and E430H and N434A and M252Y and S254T and T256E, or T307Q and Q311H and E430H and N434H and M252Y and S254T and T256E, or T307Q and Q311H and E430H and N434Y and M252Y and S254T and T256E, or T307Q and V308P and N434Y and Y436H and M252Y and S254T and T256E.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/586,679, filed May 4, 2017 which is a continuation of InternationalApplication Number PCT/EP2015/075656, filed Nov. 4, 2015, and claimsbenefit under 35 U.S.C. § 119 to European Application Number 14192052.0,filed Nov. 6, 2014, which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on May 6, 2020, is namedSequence_listing.txt and is 59,447 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies and Fc-region fusionpolypeptides which are asymmetrically modified with respect to theirFc-receptor, especially their FcRn, interaction and methods of using thesame.

BACKGROUND

The neonatal Fc-receptor (FcRn) is important for the metabolic fate ofantibodies of the IgG class in vivo. The FcRn functions to salvage IgGfrom the lysosomal degradation pathway, resulting in reduced clearanceand increased half-life. It is a heterodimeric protein consisting of twopolypeptides: a 50 kDa class I major histocompatibility complex-likeprotein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds withhigh affinity to the CH2-CH3 portion of the Fc-region of an antibody ofthe class IgG. The interaction between an antibody of the class IgG andthe FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgGantibody molecule can interact with two FcRn molecules via its two heavychain Fc-region polypeptides (see e.g. Huber, A. H., et al., J. Mol.Biol. 230 (1993) 1077-1083).

Thus, an IgGs in vitro FcRn binding properties/characteristics areindicative of its in vivo pharmacokinetic properties in the bloodcirculation.

In the interaction between the FcRn and the Fc-region of an antibody ofthe IgG class different amino acid residues of the heavy chain CH2- andCH3-domain are participating.

Different mutations that influence the FcRn binding and therewith thehalf-live in the blood circulation are known. Fc-region residuescritical to the mouse Fc-region-mouse FcRn interaction have beenidentified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., etal. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434,and H435 (numbering according to Kabat EU index numbering system) areinvolved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26(1996) 2533-2536; Firan, M., et al., Int. Immunol. 13 (2001) 993-1002;Kim, J. K., et al., Eur. J. Immunol. 24 (1994) 542-548). Residues I253,H310, and H435 were found to be critical for the interaction of humanFc-region with murine FcRn (Kim, J. K., et al., Eur. J. Immunol. 29(1999) 2819-2885).

Methods to increase Fc-region (and likewise IgG) binding to FcRn havebeen performed by mutating various amino acid residues in the Fc-region:Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433,and Asn 434 (see Kuo, T. T., et al., J. Clin. Immunol. 30 (2010)777-789; Ropeenian, D. C., et al., Nat. Rev. Immunol. 7 (2007) 715-725).

The combination of the mutations M252Y, S254T, T256E have been describedby Dall'Acqua et al. to improve FcRn binding by protein-proteininteraction studies (Dall'Acqua, W. F., et al. J. Biol. Chem. 281 (2006)23514-23524). Studies of the human Fc-region-human FcRn complex haveshown that residues I253, S254, H435, and Y436 are crucial for theinteraction (Firan, M., et al., Int. Immunol. 13 (2001) 993-1002;Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung,Y. A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants ofresidues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 havebeen reported and examined.

SUMMARY

It has been found that the FcRn-binding of an antibody or Fc-regionfusion polypeptide can be modified by altering amino acid residues atnon-corresponding positions in the individual Fc-region polypeptides asthese alterations act together in the modification of the FcRn-binding.Antibodies and Fc-region fusion polypeptides as reported herein areuseful, e.g., for the treatment of diseases in which tailor-madesystemic retention times are required.

Herein are reported variant Fc-regions that have modified FcRn bindingproperties compared to a corresponding wild-type Fc-region. Thesevariant Fc-regions contain specific amino acid mutations in the CH2-and/or CH3-domain. It has been found that these mutations when usedeither alone or in combination in the same or distributed in both heavychains of an Fc-region allow to tailor-design the in vivo half-live ofthe variant Fc-region.

One aspect as reported herein is a variant (human) IgG class Fc-regioncomprising a first Fc-region polypeptide and a second Fc-regionpolypeptide,

-   -   wherein    -   a) the first Fc-region polypeptide and the second Fc-region        polypeptide are derived from the same parent (human) IgG class        Fc-region polypeptide, and    -   b) the first Fc-region polypeptide has an amino acid sequence        that differs from the second Fc-region polypeptide amino acid        sequence at least at one corresponding position according to the        Kabat EU index numbering system,    -   whereby the variant (human) IgG class Fc-region has a different        affinity to a human Fc-receptor compared to a (human) IgG class        Fc-region that has the same amino acid residues (as the (parent)        human Fc-region polypeptide of a)) at corresponding positions        according to the Kabat EU index numbering system in the first        Fc-region polypeptide and the second Fc-region polypeptide.

One aspect as reported herein is a variant (human) IgG class Fc-regioncomprising a first Fc-region polypeptide and a second Fc-regionpolypeptide,

-   -   wherein    -   a) the first Fc-region polypeptide has an amino acid sequence        that differs from the second Fc-region polypeptide amino acid        sequence at least at one corresponding position according to the        Kabat EU index numbering system,    -   whereby the variant (human) IgG class Fc-region has a different        affinity to a human Fc-receptor compared to an IgG class        Fc-region that has the same amino acid residue (as in a        corresponding human Fc-region) in the first and the second        Fc-region polypeptide at the corresponding position.

One aspect as reported herein is a variant (human) IgG class Fc-regioncomprising a first Fc-region polypeptide and a second Fc-regionpolypeptide,

-   -   wherein    -   a) the amino acid sequence of the first Fc-region polypeptide        differs from the amino acid sequence of a first parent IgG class        Fc-region polypeptide in one or more amino acid residues,        -   and        -   the amino acid sequence of the second Fc-region polypeptide            differs from the amino acid sequence of a second parent IgG            class Fc-region polypeptide in one or more amino acid            residues, and    -   b) the first Fc-region polypeptide has an amino acid sequence        that differs from the second Fc-region polypeptide amino acid        sequence at least at one corresponding position according to the        Kabat EU index numbering system,    -   whereby the variant (human) IgG class Fc-region has a different        affinity to a human Fc-receptor compared to a parent IgG class        Fc-region comprising the first and the second parent IgG class        Fc-region polypeptide of a).

One aspect as reported herein is a variant (human) IgG class Fc-regioncomprising a first Fc-region polypeptide and a second Fc-regionpolypeptide,

-   -   wherein    -   a) the amino acid sequence of the first Fc-region polypeptide is        derived from a first parent IgG class Fc-region polypeptide and        the amino acid sequence of the second Fc-region polypeptide is        derived from a second parent IgG class Fc-region polypeptide,        and    -   b) in the first Fc-region polypeptide and/or in the second        Fc-region polypeptide one or more mutations are introduced so        that the first Fc-region polypeptide has an amino acid sequence        that differs from the second Fc-region polypeptide amino acid        sequence at least at one corresponding position according to the        Kabat EU index numbering system,    -   whereby the variant (human) IgG class Fc-region has a different        affinity to a human Fc-receptor compared to an IgG class        Fc-region comprising the first and the second parent IgG class        Fc-region polypeptide of a).

In one embodiment of all aspects the variant (human) IgG class Fc-regionis a variant (human) IgG class heterodimeric Fc-region.

In one embodiment of all aspects the first parent IgG class Fc-regionpolypeptide and the second parent IgG class Fc-region polypeptide arenon-human IgG class Fc-region polypeptides.

In one embodiment of all aspects the first parent IgG class Fc-regionpolypeptide and the second parent IgG class Fc-region polypeptide arethe same IgG class Fc-region polypeptide.

In one embodiment of all aspects the pairing of the first Fc-regionpolypeptide and the second Fc-region polypeptide to form a dimeric(functional) Fc-region results in the formation of a heterodimer.

In one embodiment of all aspects the first and the second Fc-regionpolypeptide differ independently of each other in at least one aminoacid residue from the respective parent IgG class Fc-region polypeptide.

In one embodiment of all aspects the IgG class is selected from thesubclasses IgG1, IgG2, IgG3 and IgG4.

In one embodiment of all aspects the human Fc-receptor is selected fromthe human neonatal Fc-receptor and the human Fcγ receptor.

In one embodiment of all aspects the first Fc-region polypeptide differsin 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 aminoacid residues at corresponding position according to the Kabat EU indexnumbering system from the second Fc-region polypeptide.

In one embodiment of all aspects as reported herein either the firstFc-region polypeptide or the second Fc-region polypeptide or bothFc-region polypeptides comprise one of the following mutations orcombination of mutations:

-   -   T307H, or    -   Q311H, or    -   E430H, or    -   N434H, or    -   T307H and Q311H, or    -   T307H and E430H, or    -   T307H and N434A, or    -   T307H and N434H, or    -   T307Q and Q311H, or    -   T307Q and E430H, or    -   T307Q and N434H, or    -   T307H and Q311H and E430H and N434A, or    -   T307H and Q311H and E430H and N434H, or    -   T307H and Q311H and E430H and N434Y, or    -   T307Q and Q311H and E430H and N434A, or    -   T307Q and Q311H and E430H and N434H, or    -   T307Q and Q311H and E430H and N434Y, or    -   T307Q and V308P and N434Y and Y436H, or    -   T307H and M252Y and S254T and T256E, or    -   T307Q and M252Y and S254T and T256E, or    -   Q311H and M252Y and S254T and T256E, or    -   E430H and M252Y and S254T and T256E, or    -   N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and M252Y and S254T and T256E, or    -   T307H and E430H and M252Y and S254T and T256E, or    -   T307H and N434A and M252Y and S254T and T256E, or    -   T307H and N434H and M252Y and S254T and T256E, or    -   T307Q and Q311H and M252Y and S254T and T256E, or    -   T307Q and E430H and M252Y and S254T and T256E, or    -   T307Q and N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and        T256E.

In one embodiment of all aspects as reported herein either the firstFc-region polypeptide or the second Fc-region polypeptide or bothFc-region polypeptides comprise one of the following mutations orcombination of mutations:

-   -   T307H, or    -   E430H, or    -   T307H and Q311H, or    -   T307H and E430H, or    -   T307H and N434A, or    -   T307H and N434H, or    -   T307Q and Q311H, or    -   T307Q and E430H, or    -   T307Q and N434H, or    -   T307H and Q311H and E430H and N434A, or    -   T307H and Q311H and E430H and N434H, or    -   T307H and Q311H and E430H and N434Y, or    -   T307Q and Q311H and E430H and N434A, or    -   T307Q and Q311H and E430H and N434H, or    -   T307Q and Q311H and E430H and N434Y, or    -   T307Q and V308P and N434Y and Y436H, or    -   T307H and M252Y and S254T and T256E, or    -   T307Q and M252Y and S254T and T256E, or    -   Q311H and M252Y and S254T and T256E, or    -   E430H and M252Y and S254T and T256E, or    -   N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and M252Y and S254T and T256E, or    -   T307H and E430H and M252Y and S254T and T256E, or    -   T307H and N434A and M252Y and S254T and T256E, or    -   T307H and N434H and M252Y and S254T and T256E, or    -   T307Q and Q311H and M252Y and S254T and T256E, or    -   T307Q and E430H and M252Y and S254T and T256E, or    -   T307Q and N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and        T256E.

In one embodiment of all aspects as reported herein

-   -   the first Fc-region polypeptide comprise independently of the        second Fc-region polypeptide one of the following mutations or        combination of mutations:        -   T307H, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E, or        -   T307H and Q311H and M252Y and S254T and T256E, or        -   T307H and E430H and M252Y and S254T and T256E, or        -   T307H and N434A and M252Y and S254T and T256E, or        -   T307H and N434H and M252Y and S254T and T256E, or        -   T307Q and Q311H and M252Y and S254T and T256E, or        -   T307Q and E430H and M252Y and S254T and T256E, or        -   T307Q and N434H and M252Y and S254T and T256E, or        -   T307H and Q311H and E430H and N434A and M252Y and S254T and            T256E, or        -   T307H and Q311H and E430H and N434H and M252Y and S254T and            T256E, or        -   T307H and Q311H and E430H and N434Y and M252Y and S254T and            T256E, or        -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and            T256E, or        -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and            T256E,    -   and    -   the second Fc-region polypeptide comprise independently of the        first Fc-region polypeptide one of the following mutations or        combination of mutations        -   T307H, or        -   T307Q, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E.

In one embodiment of all aspects as reported herein

-   -   the first Fc-region polypeptide comprise independently of the        second Fc-region polypeptide one of the following mutations or        combination of mutations:        -   T307H, or        -   E430H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E, or        -   T307H and Q311H and M252Y and S254T and T256E, or        -   T307H and E430H and M252Y and S254T and T256E, or        -   T307H and N434A and M252Y and S254T and T256E, or        -   T307H and N434H and M252Y and S254T and T256E, or        -   T307Q and Q311H and M252Y and S254T and T256E, or        -   T307Q and E430H and M252Y and S254T and T256E, or        -   T307Q and N434H and M252Y and S254T and T256E, or        -   T307H and Q311H and E430H and N434A and M252Y and S254T and            T256E, or        -   T307H and Q311H and E430H and N434H and M252Y and S254T and            T256E, or        -   T307H and Q311H and E430H and N434Y and M252Y and S254T and            T256E, or        -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and            T256E, or        -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and            T256E,    -   and    -   the second Fc-region polypeptide comprise independently of the        first Fc-region polypeptide one of the following mutations or        combination of mutations        -   T307H, or        -   T307Q, or        -   E430H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E.

In one embodiment of all aspects as reported herein the first Fc-regionpolypeptide comprises

-   -   one of the following combinations of mutations:        -   none, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A,    -   and    -   one of the following mutations or combination of mutations:        -   none        -   T307H, or        -   T307Q, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307Q and N434A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H,    -   and the second Fc-region polypeptide comprises        -   one of the following mutations or combination of mutations:            -   none, if the first Fc-region polypeptide comprises at                least one mutation, or            -   T307H, or            -   T307Q, if the first Fc-region polypeptide does not                comprises solely the T307Q mutation, or            -   Q311H, or            -   E430H, or            -   N434H, or            -   T307H and Q311H, or            -   T307H and E430H, or            -   T307H and N434A, or            -   T307H and N434H, or            -   T307Q and Q311H, or            -   T307Q and E430H, or            -   T307Q and N434H, or            -   T307Q and N434A, or            -   M252Y and S254T and T256E, if the first Fc-region                polypeptide does not comprises solely the combination                M252Y and S254T and T256E of mutations, or            -   I253A and H310A and H435A, if the first Fc-region                polypeptide does not comprises solely the combination                I253A and H310A and H435A of mutations, or            -   H310A and H433A and Y436A, if the first Fc-region                polypeptide does not comprises solely the combination                H310A and H433A and Y436A of mutations, or            -   T307H and Q311H and E430H and N434A, or            -   T307H and Q311H and E430H and N434H, or            -   T307H and Q311H and E430H and N434Y, or            -   T307Q and Q311H and E430H and N434H, or            -   T307Q and Q311H and E430H and N434Y, or            -   T307Q and V308P and N434Y and Y436H.

In one embodiment of all aspects as reported herein the first Fc-regionpolypeptide comprises

-   -   one of the following combinations of mutations:        -   none, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A,    -   and    -   one of the following mutations or combination of mutations:        -   none        -   T307H, or        -   T307Q, or        -   E430H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307Q and N434A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H,    -   and the second Fc-region polypeptide comprises        -   one of the following mutations or combination of mutations:            -   none, if the first Fc-region polypeptide comprises at                least one mutation, or            -   T307H, or            -   T307Q, if the first Fc-region polypeptide does not                comprises solely the T307Q mutation, or            -   E430H, or            -   T307H and Q311H, or            -   T307H and E430H, or            -   T307H and N434A, or            -   T307H and N434H, or            -   T307Q and Q311H, or            -   T307Q and E430H, or            -   T307Q and N434H, or            -   T307Q and N434A, or            -   M252Y and S254T and T256E, if the first Fc-region                polypeptide does not comprises solely the combination                M252Y and S254T and T256E of mutations, or            -   I253A and H310A and H435A, if the first Fc-region                polypeptide does not comprises solely the combination                I253A and H310A and H435A of mutations, or            -   H310A and H433A and Y436A, if the first Fc-region                polypeptide does not comprises solely the combination                H310A and H433A and Y436A of mutations, or            -   T307H and Q311H and E430H and N434A, or            -   T307H and Q311H and E430H and N434H, or            -   T307H and Q311H and E430H and N434Y, or            -   T307Q and Q311H and E430H and N434A, or            -   T307Q and Q311H and E430H and N434H, or            -   T307Q and Q311H and E430H and N434Y, or            -   T307Q and V308P and N434Y and Y436H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations I253A and H310A and H435A and the second Fc-regionpolypeptide comprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations I253A and H310A and H435A and the second Fc-regionpolypeptide comprises the mutations M252Y and S254T and T256E and T307Qand N434Y.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations I253A and H310A and H435A and the second Fc-regionpolypeptide comprises the mutations M252Y and S254T and T256E and T307Qand V308P and N434Y and Y436H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and Q311H and E430H and N434H and the secondFc-region polypeptide comprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307Q and N434A and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T250Q and M428L and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307Q and N434H and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E and T307Q and N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and Q311H and E430H and N434H and the secondFc-region polypeptide comprises the mutations T307H and Q311H and E430Hand N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and the second Fc-region polypeptidecomprises the mutations T307H and N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and M252Y and S254T and T256E and thesecond Fc-region polypeptide comprises the mutations T307H and N434H andM252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutation N434H and the second Fc-region polypeptide comprises themutation N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307Q and N434A.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutation N434H.

In one embodiment the first Fc-region polypeptide further comprises themutations Y349C, T366S, L368A and Y407V (“hole Fc-region polypeptide”)and the second Fc-region polypeptide further comprises the mutationsS354C and T366W (“knob Fc-region polypeptide”).

In one embodiment of all aspects the variant (human) IgG class Fc-regioncomprises a first and a second Fc-region polypeptide of human IgG1subclass wherein

-   -   a) the first and the second Fc-region polypeptide both further        comprise the mutations L234A and L235A (numbering according to        Kabat EU index numbering system), or    -   b) the first and the second Fc-region polypeptide both further        comprise the mutation P329G (numbering according to Kabat EU        index numbering system), or    -   c) the first and the second Fc-region polypeptide both further        comprise the mutations L234A and L235A and P329G (numbering        according to Kabat EU index numbering system), or    -   d) the first and the second Fc-region polypeptide both further        comprise the mutations L234A and L235A (numbering according to        Kabat EU index numbering system) and the first Fc-region        polypeptide further comprises the mutation Y349C or S354C and        the mutation T366W and the second Fc-region polypeptide further        comprises the mutation Y349C or S354C and the mutations T366S,        L368A and Y407V, or    -   e) the first and the second Fc-region polypeptide both further        comprise the mutations L234A and L235A and P329G (numbering        according to Kabat EU index numbering system) and the first        Fc-region polypeptide further comprises the mutation Y349C or        S354C and the mutation T366W and the second Fc-region        polypeptide further comprises the mutation Y349C or S354C and        the mutations T366S, L368A and Y407V.

In one embodiment the variant (human) IgG class Fc-region comprises afirst and a second Fc-region polypeptide of human IgG4 subclass wherein

-   -   a) the first and the second Fc-region polypeptide both further        comprise the mutations S228P and L235E (numbering according to        Kabat EU index numbering system), or    -   b) the first and the second Fc-region polypeptide both further        comprise the mutation P329G (numbering according to Kabat EU        index numbering system), or    -   c) the first and the second Fc-region polypeptide both further        comprise the mutations S228P and L235E and P329G (numbering        according to Kabat EU index numbering system), or    -   d) the first and the second Fc-region polypeptide both further        comprise the mutations S228P and L235E (numbering according to        Kabat EU index numbering system) and the first Fc-region        polypeptide further comprises the mutation Y349C or S354C and        the mutation T366W and the second Fc-region polypeptide further        comprises the mutation Y349C or S354C and the mutations T366S,        L368A and Y407V,    -   e) the first and the second Fc-region polypeptide both further        comprise the mutations S228P and L235E and P329G (numbering        according to Kabat EU index numbering system) and the first        Fc-region polypeptide further comprises the mutation Y349C or        S354C and the mutation T366W and the second Fc-region        polypeptide further comprises the mutation Y349C or S354C and        the mutations T366S, L368A and Y407V.

One aspect as reported herein is an antibody or Fc-region fusionpolypeptide comprising the variant (human) IgG class Fc-region asreported herein.

In one embodiment the antibody is a monoclonal antibody.

In one embodiment the antibody is a human, humanized, or chimericantibody.

One aspect as reported herein is a nucleic acid encoding the variant(human) IgG class Fc-region as reported herein.

One aspect as reported herein is a nucleic acid encoding the antibody asreported herein.

One aspect as reported herein is a nucleic acid encoding the Fc-regionfusion polypeptide as reported herein.

One aspect as reported herein is a host cell comprising the nucleic acidas reported herein.

One aspect as reported herein is a method of producing the variant(human) IgG class Fc-region as reported herein comprising culturing thehost cell as reported herein so that the variant (human) IgG classFc-region is produced.

One aspect as reported herein is a method of producing the antibody asreported herein comprising culturing the host cell as reported herein sothat the antibody is produced.

One aspect as reported herein is a method of producing the Fc-regionfusion polypeptide as reported herein comprising culturing the host cellas reported herein so that the Fc-region fusion polypeptide is produced.

One aspect as reported herein is a pharmaceutical formulation comprisingthe variant (human) IgG class Fc-region as reported herein or theantibody as reported herein or the Fc-region fusion polypeptide asreported herein.

One aspect as reported herein is the variant (human) IgG class Fc-regionas reported herein or the antibody as reported herein or the Fc-regionfusion polypeptide as reported herein for use as a medicament.

One aspect as reported herein is the use of the variant (human) IgGclass Fc-region as reported herein or the antibody as reported herein orthe Fc-region fusion polypeptide as reported herein in the manufactureof a medicament.

The antibodies as reported herein can be used as e.g. T-cell recruiters,as Fc gamma receptor binder with high biological activity (potency) andfast clearance from the blood circulation (blood serum), asantibody-drug-conjugates with fast clearance in order to reduce systemicside effects, or as pre-targeting antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Illustrative schematic FcRn affinity chromatography elutiondiagram of an antibody comprising different (variant) Fc-regions: 1:I253A/H310A/H435A mutation; 2: wild-type Fc-region; 3: M252Y/S254T/T256Ein one Fc-region polypeptide, the other wild-type Fc-region; 4:M252Y/S254T/T256E in both Fc-region polypeptides; 5: knob-chain:M252Y/S254T/T256E, hole-chain: T307Q/N434A.

FIG. 2 FcRn affinity chromatography elution diagram of an antibodycomprising different (variant) Fc-regions: 1: wild-type Fc-region; 2:glycoengineered Fc-region; 3: T307Q/N434A; 4: T307H/N434H; 5:T307H/N434H/M252Y/S254T/T256E; 6: N434H; 7: M252Y/S254T/T256E.

FIG. 3 FcRn affinity chromatography elution diagram of an antibodycomprising different (variant) Fc-regions: 1: hole chain-knob chainFc-region; 2: hole-chain: T307Q/N434A, knob-chain: M252Y/S254T/T256E; 3:hole-chain: T307H/N434H, knob-chain: M252Y/S254T/T256E; 4: hole-chain:T250Q/M428L, knob-chain: M252Y/S254T/T256E; 5: hole-chain: T307Q, N434H,knob-chain: M252Y/S254T/T256E/T307Q/N434H; 6: hole-chain:T307H/Q311H/E430H/N434H, knob-chain: M252Y/S254T/T256E; 7: hole-chain:T307H/Q311H/E430H/N434H, knob-chain:M252Y/S254T/T256E/T307H/Q311H/E430H/N434H; 8: hole-/knob-chain:M252Y/S254T/T256E.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “about” denotes a range of +/−20% of the thereafter followingnumerical value. In one embodiment the term about denotes a range of+/−10% of the thereafter following numerical value. In one embodimentthe term about denotes a range of +/−5% of the thereafter followingnumerical value.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencealterations. In some embodiments, the number of amino acid alterationsare 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4or less, 3 or less, or 2 or less. In some embodiments, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “alteration” denotes the mutation (substitution), insertion(addition), or deletion of one or more amino acid residues in a parentantibody or fusion polypeptide, e.g. a fusion polypeptide comprising atleast an FcRn binding portion of an Fc-region, to obtain a modifiedantibody or fusion polypeptide. The term “mutation” denotes that thespecified amino acid residue is substituted for a different amino acidresidue. For example the mutation L234A denotes that the amino acidresidue lysine at position 234 in an antibody Fc-region (polypeptide) issubstituted by the amino acid residue alanine (substitution of lysinewith alanine) (numbering according to the EU index).

The term “amino acid mutation” denotes the substitution of at least oneexisting amino acid residue with another different amino acid residue(=replacing amino acid residue). The replacing amino acid residue may bea “naturally occurring amino acid residues” and selected from the groupconsisting of alanine (three letter code: ala, one letter code: A),arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G),histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys,K), methionine (met, M), phenylalanine (phe, F), proline (pro, P),serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr,Y), and valine (val, V). The replacing amino acid residue may be a“non-naturally occurring amino acid residue”. See e.g. U.S. Pat. No.6,586,207, WO 98/48032, WO 03/073238, US 2004/0214988, WO 2005/35727, WO2005/74524, Chin, J. W., et al., J. Am. Chem. Soc. 124 (2002) 9026-9027;Chin, J. W. and Schultz, P. G., ChemBioChem 11 (2002) 1135-1137; Chin,J. W., et al., PICAS United States of America 99 (2002) 11020-11024;and, Wang, L. and Schultz, P. G., Chem. (2002) 1-10 (all entirelyincorporated by reference herein).

The term “amino acid insertion” denotes the (additional) incorporationof at least one amino acid residue at a predetermined position in anamino acid sequence. In one embodiment the insertion will be theinsertion of one or two amino acid residues. The inserted amino acidresidue(s) can be any naturally occurring or non-naturally occurringamino acid residue.

The term “amino acid deletion” denotes the removal of at least one aminoacid residue at a predetermined position in an amino acid sequence.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, multispecific antibodies (e.g. bispecific antibodies,trispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen- and/or protein A and/or FcRn-binding activity.

The term “asymmetric Fc-region” denotes a pair of Fc-region polypeptidesthat have different amino acid residues at corresponding positionsaccording to the Kabat EU index numbering system.

The term “asymmetric Fc-region with respect to FcRn binding” denotes anFc-region that consists of two polypeptide chains that have differentamino acid residues at corresponding positions, whereby the positionsare determined according to the Kabat EU index numbering system, wherebythe different positions affect the binding of the Fc-region to the humanneonatal Fc-receptor (FcRn). For the purpose herein the differencesbetween the two polypeptide chains of the Fc-region in an “asymmetricFc-region with respect to FcRn binding” do not include differences thathave been introduced to facilitate the formation of heterodimericFc-regions, e.g. for the production of bispecific antibodies. Thesedifferences can also be asymmetric, i.e. the two chains have differencesat non corresponding amino acid residues according to the Kabat EU indexnumbering system. These differences facilitate heterodimerization andreduce homodimerization. Examples of such differences are the so-called“knobs into holes” substitutions (see, e.g., U.S. Pat. No. 7,695,936 andUS 2003/0078385). The following knobs and holes substitutions in theindividual polypeptide chains of an Fc-region of an IgG antibody ofsubclass IgG1 have been found to increase heterodimer formation: 1)Y407T in one chain and T366Y in the other chain; 2) Y407A in one chainand T366W in the other chain; 3) F405A in one chain and T394W in theother chain; 4) F405W in one chain and T394S in the other chain; 5)Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A inone chain and T394W and Y407T in the other chain; 7) T366W and F405W inone chain and T394S and Y407A in the other chain; 8) F405W and Y407A inone chain and T366W and T394S in the other chain; and 9) T366W in onechain and T366S, L368A, and Y407V in the other chain, whereby the lastlisted is especially suited. In addition, changes creating new disulfidebridges between the two Fc-region polypeptide chains facilitateheterodimer formation (see, e.g., US 2003/0078385). The followingsubstitutions resulting in appropriately spaced apart cysteine residuesfor the formation of new intra-chain disulfide bonds in the individualpolypeptide chains of an Fc-region of an IgG antibody of subclass IgG1have been found to increase heterodimer formation: Y349C in one chainand S354C in the other; Y349C in one chain and E356C in the other; Y349Cin one chain and E357C in the other; L351C in one chain and S354C in theother; T394C in one chain and E397C in the other; or D399C in one chainand K392C in the other. Further examples of heterodimerizationfacilitating amino acid changes are the so-called “charge pairsubstitutions” (see, e.g., WO 2009/089004). The following charge pairsubstitutions in the individual polypeptide chains of an Fc-region of anIgG antibody of subclass IgG1 have been found to increase heterodimerformation: 1) K409D or K409E in one chain and D399K or D399R in theother chain; 2) K392D or K392E in one chain and D399K or D399R in theother chain; 3) K439D or K439E in one chain and E356K or E356R in theother chain; 4) K370D or K370E in one chain and E357K or E357R in theother chain; 5) K409D and K360D in one chain plus D399K and E356K in theother chain; 6) K409D and K370D in one chain plus D399K and E357K in theother chain; 7) K409D and K392D in one chain plus D399K, E356K, andE357K in the other chain; 8) K409D and K392D in one chain and D399K inthe other chain; 9) K409D and K392D in one chain and D399K and E356K inthe other chain; 10) K409D and K392D in one chain and D399K and D357K inthe other chain; 11) K409D and K370D in one chain and D399K and D357K inthe other chain; 12) D399K in one chain and K409D and K360D in the otherchain; and 13) K409D and K439D in one chain and D399K and E356K on theother.

The term “binding (to an antigen)” denotes the binding of an antibody toits antigen in an in vitro assay, in one embodiment in a binding assayin which the antibody is bound to a surface and binding of the antigento the antibody is measured by Surface Plasmon Resonance (SPR). Bindingmeans a binding affinity (K_(D)) of 10⁻⁸ M or less, in some embodimentsof 10⁻¹³ to 10⁻⁸ M, in some embodiments of 10⁻¹³ to 10⁻⁹ M.

Binding can be investigated by a BIAcore assay (GE Healthcare BiosensorAB, Uppsala, Sweden). The affinity of the binding is defined by theterms k_(a) (rate constant for the association of the antibody from theantibody/antigen complex), k_(d) (dissociation constant), andK_(D)(k_(d)/k_(a)).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The term “CH2-domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 231 to EUposition 340 (EU numbering system according to Kabat). In one embodimenta CH2 domain has the amino acid sequence of SEQ ID NO: 01: APELLGGPSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQ ESTYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.

The term “CH3-domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 341 to EUposition 446. In one embodiment the CH3 domain has the amino acidsequence of SEQ ID NO: 2: GQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYTQKSLSLSPG.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “comparable length” denotes that two polypeptides comprise theidentical number of amino acid residues or can be different in length byone or more and up to 10 amino acid residues at most. In one embodimentthe Fc-region polypeptides comprise the identical number of amino acidresidues or differ by a number of from 1 to 10 amino acid residues. Inone embodiment the Fc-region polypeptides comprise the identical numberof amino acid residues or differ by a number of from 1 to 5 amino acidresidues. In one embodiment the Fc-region polypeptides comprise theidentical number of amino acid residues or differ by a number of from 1to 3 amino acid residues.

The term “derived from” denotes that an amino acid sequence is derivedfrom a parent amino acid sequence by introducing alterations at at leastone position. Thus a derived amino acid sequence differs from thecorresponding parent amino acid sequence at at least one correspondingposition (numbering according to Kabat EU index numbering system forantibody Fc-regions). In one embodiment an amino acid sequence derivedfrom a parent amino acid sequence differs by one to fifteen amino acidresidues at corresponding positions. In one embodiment an amino acidsequence derived from a parent amino acid sequence differs by one to tenamino acid residues at corresponding positions. In one embodiment anamino acid sequence derived from a parent amino acid sequence differs byone to six amino acid residues at corresponding positions. Likewise aderived amino acid sequence has a high amino acid sequence identity toits parent amino acid sequence. In one embodiment an amino acid sequencederived from a parent amino acid sequence has 80% or more amino acidsequence identity. In one embodiment an amino acid sequence derived froma parent amino acid sequence has 90% or more amino acid sequenceidentity. In one embodiment an amino acid sequence derived from a parentamino acid sequence has 95% or more amino acid sequence identity.

“Effector functions” refer to those biological activities attributableto the Fc-region of an antibody, which vary with the antibody class.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B-cell receptor); and B-cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc-fusion polypeptide” denotes a fusion of a binding domain(e.g. an antigen binding domain such as a single chain antibody, or apolypeptide such as a ligand of a receptor) with an antibody Fc-regionthat exhibits the desired target- and/or protein A and/or FcRn-bindingactivity.

The term “Fc-region of human origin” denotes the C-terminal region of animmunoglobulin heavy chain of human origin that contains at least a partof the hinge region, the CH2 domain and the CH3 domain. In oneembodiment, a human IgG heavy chain Fc-region extends from Cys226, orfrom Pro230, to the carboxyl-terminus of the heavy chain. In oneembodiment the Fc-region has the amino acid sequence of SEQ ID NO: 60.However, the C-terminal lysine (Lys447) of the Fc-region may or may notbe present. Unless otherwise specified herein, numbering of amino acidresidues in the Fc-region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991), NIH Publication 91 3242. The Fc-region is composed of two heavychain Fc-region polypeptides, which can be covalently linked to eachother via the hinge region cysteine residues forming inter-polypeptidedisulfide bonds.

The term “FcRn” denotes the human neonatal Fc-receptor. FcRn functionsto salvage IgG from the lysosomal degradation pathway, resulting inreduced clearance and increased half-life. The FcRn is a heterodimericprotein consisting of two polypeptides: a 50 kDa class I majorhistocompatibility complex-like protein (α-FcRn) and a 15 kDaβ2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3portion of the Fc-region of IgG. The interaction between IgG and FcRn isstrictly pH dependent and occurs in a 1:2 stoichiometry, with one IgGbinding to two FcRn molecules via its two heavy chains (Huber, A. H., etal., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in theendosome at acidic pH (pH <6.5) and IgG is released at the neutral cellsurface (pH of about 7.4). The pH-sensitive nature of the interactionfacilitates the FcRn-mediated protection of IgGs pinocytosed into cellsfrom intracellular degradation by binding to the receptor within theacidic environment of endosomes. FcRn then facilitates the recycling ofIgG to the cell surface and subsequent release into the blood streamupon exposure of the FcRn-IgG complex to the neutral pH environmentoutside the cell.

The term “FcRn binding portion of an Fc-region” denotes the part of anantibody heavy chain polypeptide that extends approximately from EUposition 243 to EU position 261 and approximately from EU position 275to EU position 293 and approximately from EU position 302 to EU position319 and approximately from EU position 336 to EU position 348 andapproximately from EU position 367 to EU position 393 and EU position408 and approximately from EU position 424 to EU position 440. In oneembodiment one or more of the following amino acid residues according tothe EU numbering of Kabat are altered F243, P244, P245 P, K246, P247,K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259,T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285,N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305,L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317,E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345,P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378,V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391,T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433,N434, H435, Y436, T437, Q438, K439, and S440 (EU numbering).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “full length antibody” denotes an antibody having a structuresubstantially similar to a native antibody structure or having heavychains that contain an Fc-region as defined herein. A full lengthantibody may comprise further domains, such as e.g. a scFv or a scFabconjugated to one or more of the chains of the full length antibody.These conjugates are also encompassed by the term full length antibody.

The terms “heterodimer” or “heterodimeric” denote a molecule thatcomprises two polypeptide chains (e.g. of comparable length), whereinthe two polypeptide chains have an amino acid sequence that have atleast one different amino acid residue in a corresponding position,whereby corresponding position is determined according to the EU indexof Kabat.

The terms “homodimer” and “homodimeric” denote a molecule that comprisestwo polypeptide chains of comparable length, wherein the two polypeptidechains have an amino acid sequence that is identical in correspondingpositions, whereby corresponding positions are determined according tothe EU index of Kabat.

An antibody or Fc-region fusion polypeptide as reported herein can behomodimeric or heterodimeric with respect to its Fc-region which isdetermined with respect to mutations or properties in focus. Forexample, with respect to FcRn and/or protein A binding (i.e. the focusedon properties) an Fc-region (antibody) is homodimeric (i.e. both heavychain Fc-region polypeptides comprise these mutations) with respect tothe mutations H310A, H433A and Y436A (these mutations are in focus withrespect to FcRn and/or protein A binding property of the Fc-regionfusion polypeptide or antibody) but at the same time heterodimeric withrespect to the mutations Y349C, T366S, L368A and Y407V (these mutationsare not in focus as these mutations are directed to theheterodimerization of the heavy chains and not to the FcRn/protein Abinding properties) as well as the mutations S354C and T366W,respectively (the first set is comprised only in the first Fc-regionpolypeptide whereas the second set is comprised only in the secondFc-region polypeptide). Further for example, an Fc-region fusionpolypeptide or an antibody as reported herein can be heterodimeric withrespect to the mutations I253A, H310A, H433A, H435A and Y436A (i.e.these mutations are directed all to the FcRn and/or protein A bindingproperties of the dimeric polypeptide), i.e. one Fc-region polypeptidecomprises the mutations I253A, H310A and H435A, whereas the otherFc-region polypeptide comprises the mutations H310A, H433A and Y436A.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

The term “human Fc-region polypeptide” denotes an amino acid sequencewhich is identical to a “native” or “wild-type” human Fc-regionpolypeptide. The term “variant (human) Fc-region polypeptide” denotes anamino acid sequence which derived from a “native” or “wild-type” humanFc-region polypeptide by virtue of at least one “amino acid alteration”.A “human Fc-region” is consisting of two human Fc-region polypeptides. A“variant (human) Fc-region” is consisting of two Fc-region polypeptides,whereby both can be variant (human) Fc-region polypeptides or one is ahuman Fc-region polypeptide and the other is a variant (human) Fc-regionpolypeptide.

In one embodiment the human Fc-region polypeptide has the amino acidsequence of a human IgG1 Fc-region polypeptide of SEQ ID NO: 03, or of ahuman IgG2 Fc-region polypeptide of SEQ ID NO: 04, or of a human IgG3Fc-region polypeptide of SEQ ID NO: 05, or of a human IgG4 Fc-regionpolypeptide of SEQ ID NO: 06. In one embodiment the variant (human)Fc-region polypeptide is derived from an Fc-region polypeptide of SEQ IDNO: 03, or 04, or 05, or 06 and has at least one amino acid mutationcompared to the human Fc-region polypeptide of SEQ ID NO: 03, or 04, or05, or 06. In one embodiment the variant (human) Fc-region polypeptidecomprises/has from about one to about twelve amino acid mutations, andin one embodiment from about one to about eight amino acid mutations. Inone embodiment the variant (human) Fc-region polypeptide has at leastabout 80% homology with a human Fc-region polypeptide of SEQ ID NO: 03,or 04, or 05, or 06. In one embodiment the variant (human) Fc-regionpolypeptide has least about 90% homology with a human Fc-regionpolypeptide of SEQ ID NO: 03, or 04, or 05, or 06. In one embodiment thevariant (human) Fc-region polypeptide has at least about 95% homologywith a human Fc-region polypeptide of SEQ ID NO: 03, or 04, or 05, or06.

The variant (human) Fc-region polypeptide derived from a human Fc-regionpolypeptide of SEQ ID NO: 03, or 04, or 05, or 06 is defined by theamino acid alterations that are contained. Thus, for example, the termP329G denotes a variant (human) Fc-region polypeptide derived humanFc-region polypeptide with the mutation of proline to glycine at aminoacid position 329 relative to the human Fc-region polypeptide of SEQ IDNO: 03, or 04, or 05, or 06.

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” herein. Specifically the Kabat numberingsystem (see pages 647-660) of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) is used for the light chainconstant domain CL of kappa and lambda isotype and the Kabat EU indexnumbering system (see pages 661-723) is used for the constant heavychain domains (CH1, Hinge, CH2 and CH3).

A human IgG1 Fc-region polypeptide has the following amino acidsequence:

(SEQ ID NO: 03) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with the mutationsL234A, L235A has the following amino acid sequence:

(SEQ ID NO: 07) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with Y349C, T366S,L368A and Y407V mutations has the following amino acid sequence:

(SEQ ID NO: 08) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with S354C, T366Wmutations has the following amino acid sequence:

(SEQ ID NO: 09) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235Amutations and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 10) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a L234A, L235Aand S354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 11) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P329Gmutation has the following amino acid sequence:

(SEQ ID NO: 12) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235Amutations and P329G mutation has the following amino acid sequence:

(SEQ ID NO: 13) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P239Gmutation and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 14) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P329Gmutation and S354C, T366W mutation has the following amino acidsequence:

(SEQ ID NO: 15) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A,P329G and Y349C, T366S, L368A, Y407V mutations has the following aminoacid sequence:

(SEQ ID NO: 16) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A,P329G mutations and S354C, T366W mutations has the following amino acidsequence:

(SEQ ID NO: 17) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG4 Fc-region polypeptide has the following amino acidsequence:

(SEQ ID NO: 06) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S228P andL235E mutations has the following amino acid sequence:

(SEQ ID NO: 18) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S228P, L235Emutations and P329G mutation has the following amino acid sequence:

(SEQ ID NO: 19) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S354C, T366Wmutations has the following amino acid sequence:

(SEQ ID NO: 20) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with Y349C, T366S,L368A, Y407V mutations has the following amino acid sequence:

(SEQ ID NO: 21) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235Eand S354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 22) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235Eand Y349C, T366S, L368A, Y407V mutations has the following amino acidsequence:

(SEQ ID NO: 23) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P329Gmutation has the following amino acid sequence:

(SEQ ID NO: 24) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P239G andY349C, T366S, L368A, Y407V mutations has the following amino acidsequence:

(SEQ ID NO: 25) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P329G andS354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 26) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P,L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 27) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P,L235E, P329G and S354C, T366W mutations has the following amino acidsequence:

(SEQ ID NO: 28) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., the CDRs)correspond to those of a non-human antibody, and all or substantiallyall of the FRs correspond to those of a human antibody. A humanizedantibody optionally may comprise at least a portion of an antibodyconstant region derived from a human antibody. A “humanized form” of anantibody, e.g., a non-human antibody, refers to an antibody that hasundergone humanization.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and formstructurally defined loops (“hypervariable loops”), and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). HVRs as denoted herein include

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987)        901-917);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat, E. A. et al., Sequences of Proteins of Immunological        Interest, 5th ed. Public Health Service, National Institutes of        Health, Bethesda, Md. (1991), NIH Publication 91-3242.);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according to theKabat EU index numbering system (Kabat et al., supra).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., size-exclusion chromatographyor ion exchange or reverse phase HPLC). For review of methods forassessment of antibody purity, see, e.g., Flatman, S. et al., J. Chrom.B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “peptidic linker” as used herein denotes a peptide with aminoacid sequences, which is in one embodiment of synthetic origin. Thepeptidic linker is in one embodiment a peptide with an amino acidsequence with a length of at least 30 amino acids, in one embodimentwith a length of 32 to 50 amino acids. In one embodiment the peptidiclinker is a peptide with an amino acid sequence with a length of 32 to40 amino acids. In one embodiment the peptidic linker is (GxS)n withG=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8), inone embodiment with x=4, n=6 or 7, in one embodiment with x=4, n=7. Inone embodiment the peptidic linker is (G₄S)₆G₂.

The term “recombinant antibody” denotes all antibodies (chimeric,humanized and human) that are prepared, expressed, created or isolatedby recombinant means. This includes antibodies isolated from a host cellsuch as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant antibodies have variable and constant regions in arearranged form. The recombinant antibodies as reported herein can besubjected to in vivo somatic hypermutation. Thus, the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germ line VH andVL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies orFc-region fusion polypeptides as reported herein are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in a (antibody)molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent”denote the presence of two binding site, four binding sites, and sixbinding sites, respectively, in a (antibody) molecule. The bispecificantibodies as reported herein are in one preferred embodiment“bivalent”.

The term “variable region” or “variable domain” refer to the domain ofan antibody heavy or light chain that is involved in binding of theantibody to its antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of an antibody generally havesimilar structures, with each domain comprising four framework regions(FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T. J. etal. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page91). A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “ocular vascular disease” includes, but is not limited tointraocular neovascular syndromes such as diabetic retinopathy, diabeticmacular edema, retinopathy of prematurity, neovascular glaucoma, retinalvein occlusions, central retinal vein occlusions, macular degeneration,age-related macular degeneration, retinitis pigmentosa, retinalangiomatous proliferation, macular telangectasia, ischemic retinopathy,iris neovascularization, intraocular neovascularization, cornealneovascularization, retinal neovascularization, choroidalneovascularization, and retinal degeneration (see e.g. Garner, A.,Vascular diseases, In: Pathobiology of ocular disease, A dynamicapproach, Garner, A., and Klintworth, G. K., (eds.), 2nd edition, MarcelDekker, New York (1994), pp. 1625-1710).

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The term “with (the) mutation IHH-AAA” as used herein refers to thecombination of the mutations I253A (Ile253Ala), H310A (His310Ala), andH435A (His435Ala) and the term “with (the) mutation HHY-AAA” as usedherein refers to the combination of the mutations H310A (His310Ala),H433A (His433Ala), and Y436A (Tyr436Ala) and the term “with (the)mutation YTE” as used herein refers to the combination of mutationsM252Y (Met252Tyr), S254T (Ser254Thr), and T256E (Thr256Glu) in theconstant heavy chain region of IgG1 or IgG4 subclass, wherein thenumbering is according to the EU Index of Kabat.

The term “with (the) mutations P329G LALA” as used herein refers to thecombination of the mutations L234A (Leu234Ala), L235A (Leu235Ala) andP329G (Pro329Gly) in the constant heavy chain region of IgG1 subclass,wherein the numbering is according to the EU Index of Kabat. The term“with (the) mutation SPLE” as used herein refers to the combination ofthe mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constantheavy chain region of IgG4 subclass, wherein the numbering is accordingto the EU Index of Kabat. The term “with (the) mutation SPLE and P239G”as used herein refers to the combination of the mutations S228P(Ser228Pro), L235E (Leu235Glu) and P329G (Pro329Gly) in the constantheavy chain region of IgG4 subclass, wherein the numbering is accordingto the EU Index of Kabat.

II. The Current Invention

The invention is based, at least in part, on the finding that theFcRn-binding of an antibody or Fc-region fusion polypeptide can bemodified by altering amino acid residues at non-corresponding positionsin the individual Fc-region polypeptides as these alterations acttogether in the modification of the FcRn-binding. Fc-regions, antibodiesand Fc-region fusion polypeptides as reported herein are useful, e.g.,for the treatment of diseases in which tailor-made systemic retentiontimes are required.

Herein are reported variant Fc-regions that have modified FcRn bindingproperties compared to a corresponding wild-type Fc-region. Thesevariant Fc-regions contain specific amino acid mutations in the CH2-and/or CH3-domain. It has been found that these mutations when usedeither alone or in combination in the same or in both heavy chains of anFc-region allow to tailor-design the in vivo half-live of the variantFc-region.

A. The Neonatal Fc-Receptor (FcRn)

The neonatal Fc-receptor (FcRn) is important for the metabolic fate ofantibodies of the IgG class in vivo. The FcRn functions to salvagewild-type IgG from the lysosomal degradation pathway, resulting inreduced clearance and increased half-life. It is a heterodimeric proteinconsisting of two polypeptides: a 50 kDa class I majorhistocompatibility complex-like protein (α-FcRn) and a 15 kDaβ2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3portion of the Fc-region of an antibody of the class IgG. Theinteraction between an antibody of the class IgG and the FcRn is pHdependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibodymolecule can interact with two FcRn molecules via its two heavy chainFc-region polypeptides (see e.g. Huber, A. H., et al., J. Mol. Biol. 230(1993) 1077-1083).

Thus, an IgGs in vitro FcRn binding properties/characteristics areindicative of its in vivo pharmacokinetic properties in the bloodcirculation.

In the interaction between the FcRn and the Fc-region of an antibody ofthe IgG class different amino acid residues of the heavy chain CH2- andCH3-domain are participating. The amino acid residues interacting withthe FcRn are located approximately between EU position 243 and EUposition 261, approximately between EU position 275 and EU position 293,approximately between EU position 302 and EU position 319, approximatelybetween EU position 336 and EU position 348, approximately between EUposition 367 and EU position 393, at EU position 408, and approximatelybetween EU position 424 and EU position 440. More specifically thefollowing amino acid residues according to the EU numbering of Kabat areinvolved in the interaction between the Fc-region and the FcRn: F243,P244, P245 P, K246, P247, K248, D249, T250, L251, M252, I253, S254,R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279,D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292,E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312,W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340,G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373,P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385,Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428,H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439, andS440.

Site-directed mutagenesis studies have proven that the critical bindingsites in the Fc-region of IgGs for FcRn are Histidine 310, Histidine435, and Isoleucine 253 and to a lesser extent Histidine 433 andTyrosine 436 (see e.g. Kim, J. K., et al., Eur. J. Immunol. 29 (1999)2819-2825; Raghavan, M., et al., Biochem. 34 (1995) 14649-14657;Medesan, C., et al., J. Immunol. 158 (1997) 2211-2217).

Methods to increase IgG binding to FcRn have been performed by mutatingIgG at various amino acid residues: Threonine 250, Methionine 252,Serine 254, Threonine 256, Threonine 307, Glutamic acid 380, Methionine428, Histidine 433, and Asparagine 434 (see Kuo, T. T., et al., J. Clin.Immunol. 30 (2010) 777-789).

In some cases antibodies with reduced half-life in the blood circulationare desired. For example, drugs for intravitreal application should havea long half-live in the eye and a short half-life in the circulation ofthe patient. Such antibodies also have the advantage of increasedexposure to a disease site, e.g. in the eye.

Different mutations that influence the FcRn binding and therewith thehalf-live in the blood circulation are known. Fc-region residuescritical to the mouse Fc-mouse FcRn interaction have been identified bysite-directed mutagenesis (see e.g. Dall'Acqua, W. F., et al. J. Immunol169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EUnumbering according to Kabat) are involved in the interaction (Medesan,C., et al., Eur. J. Immunol. 26 (1996) 2533-2536; Firan, M., et al.,Int. Immunol. 13 (2001) 993-1002; Kim, J. K., et al., Eur. J. Immunol.24 (1994) 542-548). Residues I253, H310, and H435 were found to becritical for the interaction of human Fc with murine FcRn (Kim, J. K.,et al., Eur. J. Immunol. 29 (1999) 2819-2825). Residues M252Y, S254T,T256E have been described by Dall'Acqua et al. to improve FcRn bindingby protein-protein interaction studies (Dall'Acqua, W. F., et al. J.Biol. Chem. 281 (2006) 23514-23524). Studies of the human Fc-human FcRncomplex have shown that residues I253, S254, H435, and Y436 are crucialfor the interaction (Firan, M., et al., Int. Immunol. 13 (2001)993-1002; Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604).In Yeung, Y. A., et al. (J. Immunol. 182 (2009) 7667-7671) variousmutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to437 have been reported and examined. Exemplary mutations and theireffect on FcRn binding are listed in the following Table.

TABLE effect on half-live FcRn in the mutation binding circulationreference H285 reduced reduced Kim, J. K., H310Q/H433N (murine) (inmouse) Scand. J. Immunol. (murine IgG1) 40 (1994) 457-465 I253A reducedreduced Ghetie, V. and H310A (murine) (in mouse) Ward, E. S., H435AImmunol. Today H436A 18 (1997) (murine IgG1) 592-598 T252L/T254S/T256Fincreased increased Ghetie, V. and T252A/T254S/T256A (murine) (in mouse)Ward, E. S., (murine IgG1) Immunol. Today 18 (1997) 592-598 I253Areduced reduced Medesan, C., H310A (murine) (in mouse) et al., H435A J.Immunol. H436A 158 (1997) H433A/N434Q 2211-2217 (murine IgG1) I253Areduced reduced Kim, J. K., H310A H310A: <0.1 (in mouse) Eur. J.Immunol. H435A rel. binding 29 (1999) H435R to muFcRn 2819-2825 (humanIgG1) (murine) H433A 1.1 rel. binding Kim, J. K., (human IgG1) tomuFcRn, Eur. J. Immunol. 0.4 rel. binding 29 (1999) hu FcRn (murine)2819-2825 I253A reduced <0.1 reduced Shields, R. L., S254A relative etal., H435A binding to J. Biol. Chem. Y436A huFcRn 276 (2001) (humanIgG1) 6591-6604 R255A reduced reduced Shields, R. L., K288A (human) etal., L309A J. Biol. Chem. S415A 276 (2001) H433A 6591-6604 (human IgG1)P238A increased increased Shields, R. L., T256A (human) et al., E272A J.Biol. Chem. V305A 276 (2001) T307A 6591-6604 Q311A D312A K317A D376AA378Q E380A E382A S424A N434A K288A/N434A E380A/N434A T307A/E380A/N434A(human IgG1) H435A reduced <0.1 reduced Firan, M., (humanized IgG1) rel.binding et al., to huFcRn Int. Immunol. 13 (2001) 993-1002 I253A (nobinding) increased reduced Dall'Acqua, M252W (murine and (in mouse) J.Immunol. M252Y human) 169 (2002) M252Y/T256Q 5171-5180 M252F/T256DN434F/Y436H M252Y/S254T/T256E G385A/Q386P/N389S H433K/N434F/Y436HH433R/N434Y/Y436H G385R/Q386T/P387R/N389P M252Y/S254T/T256E/H433K/N434F/Y436H M252Y/S254T/T256E/G385R/ Q386T/P387R/N389P (human IgG1)M428L increased increased Hinton, P. R., T250Q/M428L (human) (in monkey)et al., (human IgG2) J. Biol. Chem. 279 (2004) 6213-6216M252Y/S254T/T256E + increased increased Vaccaro, C., H433K/N434F (human)(in mouse) et al., (human IgG) Nat. Biotechnol. 23 (2005) 1283-1288T307A/E380A/N434A increased increased in Pop, L. M., (chimeric IgG1)transgenic et al., Int. mouse Immunopharmacol. 5 (2005) 1279-1290 T250Qincreased increased in Petkova, S. B., E380A (human) transgenic et al.,M428L mouse Int. Immunol N434A 18 (2006) K288A/N434A 1759-1769E380A/N434A T307A/E380A/N434A (human IgG1) I253A reduced reduced inPetkova, S. B., (human IgG1) (human) transgenic et al., mouse Int.Immunol 18 (2006) 1759-1769 S239D/A330L/I332E increased increased inDall'Acqua, M252Y/S254T/T256E (human and Cynomolgus W. F., et al.,(humanized) Cynomolgus) J. Biol. Chem. 281 (2006) 23514-23524 T250Qincreased increased in Hinton, P. R., M428L (human) Rhesus apes et al.,T250Q/M428L J. Immunol. (human IgG1) 176 (2006) 346-356 T250Q/M428Lincreased no change in Datta-Mannan, P257I/Q311I (mouse and CynomolgusA., et al., (humanized IgG1) Cynomolgus) increased in J. Biol. Chem.mouse 282 (2007) 1709-1717 P257I/Q311I increased reduced inDatta-Mannan, P257I/N434H at pH 6 mice A., et al., D376V/N434H (human,P257I/N434H Drug Metab. (humanized IgG1) Cynomolgus, reduced in Dispos.mouse) Cynomolgus 35 (2007) 86-94 abrogate FcRn binding: increased andreducing the Ropeenian, I253 reduced binding ability D. C. and H310 ofIgG for Akilesh, S., H433 FcRn reduces Nat. Rev. H435 its serum Immunol.reduce FcRn binding: persistence; a 7 (2007) Y436 higher-affinity715-725 increased FcRn binding: FcRn-IgG T250 interaction N252 prolongsthe S254 half-lives of T256 IgG and Fc- T307 coupled drugs M428 in theserum N434 N434A increased increased in Yeung, Y. A., T307Q/N434A(Cynomolgus Cynomolgus et al., T307Q/N434S monkey) monkey Cancer Res.V308P/N434A 70 (2010) T307Q/E380A/N434A 3269-3277 (human IgG1) 256Pincreased at WO 2011/ 280K neutral pH 122011 339T 385H 428L 434W/Y/F/A/H(human IgG)

It has been found that one mutation one-sided in one Fc-regionpolypeptide is sufficient to weaken the binding to an Fc receptorsignificantly. The more mutations are introduced into the Fc-region theweaker the binding to the FcRn becomes. But one-sided asymmetricmutations are not sufficient to completely inhibit FcRn binding.Mutations on both sides are necessary to completely inhibit FcRnbinding.

Thus, the variant (human) IgG class Fc-region is a heterodimer and thepairing of the first (heavy chain) Fc-region polypeptide and the second(heavy chain) Fc-region polypeptide to form a functional Fc-regionresults in the formation of a heterodimer.

The results of a symmetric engineering of an IgG1 Fc-region to influenceFcRn binding is shown in the following table (alignment of mutations andretention time on an FcRn-affinity chromatography column).

TABLE FcRn- FcRn- FcRn- FcRn- binding binding binding affinity effectorfunction influ- influ- influ- column re- influencing encing encingencing tention time mutations mutation 1 mutation 2 mutation 3 [min]L234A/L235A/P329G — — — 45.3 L234A/L235A/P329G I253A H310A H435A 2.3L234A/L235A/P329G I253A — — 2.7 L234A/L235A/P329G — H310A — 2.4L234A/L235A/P329G — — H435A 2.7 L234A/L235A/P329G I253A H310A — 2.3L234A/L235A/P329G I253A — H435A 2.3 L234A/L235A/P329G — H310A H435A 2.4L234A/L235A/P329G — H310A Y436A 2.3 L234A/L235A/P329G H310A H433A Y436A2.4 L234A/L235A/P329G — — Y436A 41.3

Retention times below 3 minutes correspond to no binding as thesubstance is in the flow-through (void peak).

The single mutation H310A is the most silent symmetrical mutation todelete any FcRn-binding.

The symmetric single mutation I253A and H435A result in a relative shiftof retention time of 0.3-0.4 min. This can be generally regarded as anon-detectable binding.

The single mutation Y436A results in detectable interaction strength tothe FcRn affinity column. Without being bound by this theory thismutation could have an FcRn mediated half-life which can bedifferentiated from a zero interaction such as the combination of theI253A, H310A and H435A mutations (IHH-AAA mutation).

The results obtained with a symmetrically modified anti-HER2 antibodyare presented in the following table (see WO 2006/031370 for reference).

TABLE retention time mutation [min] I253H no binding M252D no bindingS254D no binding R255D 41.4 M252H 43.6 K288E 45.2 L309H 45.5 E258H 45.6T256H 46.0 K290H 46.2 D98E 46.2 wild-type 46.3 K317H 46.3 Q311H 46.3E430H 46.4 T307H 47.0 N434H 52.0

The Fc-region in the Fc-region fusion polypeptide confers the abovedescribed characteristics to its fusion partner. The fusion partner canbe any molecules having a biological activity whose in vivo half-liveshall be reduced or increased, i.e. whose in vivo half-live shall beclearly defined and tailor-made for its intended application.

Fc-region fusion polypeptides may comprise e.g. a variant (human) IgGclass Fc-region as reported herein and a receptor protein that binds toa target including a ligand, such as, for example, TNFR-Fc-region fusionpolypeptide (TNFR=human tumor necrosis factor receptor), orIL-1R-Fc-region fusion polypeptide (IL-1R=human interleukin-1 receptor),or VEGFR-Fc-region fusion polypeptides (VEGFR=human vascular endothelialgrowth factor receptor), or ANG2R-Fc-region fusion polypeptides(ANG2R=human angiopoietin 2 receptor).

Fc-region fusion polypeptides may comprise e.g. a variant (human) IgGclass Fc-region as reported herein and an antibody fragment that bindsto a target including, such as, for example, an antibody Fab fragment,scFvs (see e.g. Nat. Biotechnol. 23 (2005) 1126-1136), or domainantibodies (dAbs) (see e.g. WO 2004/058821, WO 2003/002609).

Fc-region fusion polypeptides may comprise e.g. a variant (human) IgGclass Fc-region as reported herein and a receptor ligand (eithernaturally occurring or artificial).

B. Exemplary Fc-Regions and Antibodies Comprising these Fc-Regions

In one aspect, the invention provides Fc-regions that have modifiedFcRn-binding, i.e. these Fc-regions bind to human FcRn with an affinityhigher or lower than an Fc-region having no mutations affecting theFcRn-binding.

In one aspect, the invention provides isolated antibodies that havemodified FcRn-binding, i.e. these antibodies bind to human FcRn with anaffinity higher or lower than an antibody having no mutations affectingthe FcRn-binding.

One aspect as reported herein is an antibody comprising a (variant)Fc-region comprising a first Fc-region polypeptide and a secondFc-region polypeptide,

wherein

-   a) the first Fc-region polypeptide and the second Fc-region    polypeptide are derived from the same human Fc-region polypeptide,    and-   b) the first Fc-region polypeptide has been modified in that its    amino acid sequence differs from the second Fc-region polypeptide    amino acid sequence at least at one corresponding position according    to the Kabat EU index numbering system, and the second Fc-region    polypeptide has been modified in that its amino acid sequence    differs from the first Fc-region polypeptide amino acid sequence at    least at one corresponding position according to the Kabat EU index    numbering system, whereby the modified position in the first    Fc-region polypeptide and the modified position in the second    Fc-region polypeptide are different, and-   c) the Fc-region has a different affinity to a human Fc-receptor    compared to an Fc-region that comprises as first and second    Fc-region polypeptide the human Fc-region polypeptide of a) (i.e.    that has the same amino acid residues as the human Fc-region    polypeptide of a) at corresponding positions according to the Kabat    EU index numbering system).

In one embodiment of all aspects as reported herein either the firstFc-region polypeptide or the second Fc-region polypeptide or bothFc-region polypeptides comprise one of the following mutations orcombination of mutations:

-   -   T307H, or    -   Q311H, or    -   E430H, or    -   N434H, or    -   T307H and Q311H, or    -   T307H and E430H, or    -   T307H and N434A, or    -   T307H and N434H, or    -   T307Q and Q311H, or    -   T307Q and E430H, or    -   T307Q and N434H, or    -   T307H and Q311H and E430H and N434A, or    -   T307H and Q311H and E430H and N434H, or    -   T307H and Q311H and E430H and N434Y, or    -   T307Q and Q311H and E430H and N434A, or    -   T307Q and Q311H and E430H and N434H, or    -   T307Q and Q311H and E430H and N434Y, or    -   T307Q and V308P and N434Y and Y436H, or    -   T307H and M252Y and S254T and T256E, or    -   T307Q and M252Y and S254T and T256E, or    -   Q311H and M252Y and S254T and T256E, or    -   E430H and M252Y and S254T and T256E, or    -   N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and M252Y and S254T and T256E, or    -   T307H and E430H and M252Y and S254T and T256E, or    -   T307H and N434A and M252Y and S254T and T256E, or    -   T307H and N434H and M252Y and S254T and T256E, or    -   T307Q and Q311H and M252Y and S254T and T256E, or    -   T307Q and E430H and M252Y and S254T and T256E, or    -   T307Q and N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and        T256E.

In one embodiment of all aspects as reported herein

-   -   the first Fc-region polypeptide comprise independently of the        second Fc-region polypeptide one of the following mutations or        combination of mutations:        -   T307H, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E, or        -   T307H and Q311H and M252Y and S254T and T256E, or        -   T307H and E430H and M252Y and S254T and T256E, or        -   T307H and N434A and M252Y and S254T and T256E, or        -   T307H and N434H and M252Y and S254T and T256E, or        -   T307Q and Q311H and M252Y and S254T and T256E, or        -   T307Q and E430H and M252Y and S254T and T256E, or        -   T307Q and N434H and M252Y and S254T and T256E, or        -   T307H and Q311H and E430H and N434A and M252Y and S254T and            T256E, or        -   T307H and Q311H and E430H and N434H and M252Y and S254T and            T256E, or        -   T307H and Q311H and E430H and N434Y and M252Y and S254T and            T256E, or        -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and            T256E, or        -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and            T256E,    -   and    -   the second Fc-region polypeptide comprise independently of the        first Fc-region polypeptide one of the following mutations or        combination of mutations        -   T307H, or        -   T307Q, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E.

In one embodiment of all aspects as reported herein the first Fc-regionpolypeptide comprises

-   -   one of the following combinations of mutations:        -   none, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A,    -   and    -   one of the following mutations or combination of mutations:        -   none        -   T307H, or        -   T307Q, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307Q and N434A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H,    -   and the second Fc-region polypeptide comprises        -   one of the following mutations or combination of mutations:            -   none, if the first Fc-region polypeptide comprises at                least one mutation, or            -   T307H, or            -   T307Q, if the first Fc-region polypeptide does not                comprises solely the T307Q mutation, or            -   Q311H, or            -   E430H, or            -   N434H, or            -   T307H and Q311H, or            -   T307H and E430H, or            -   T307H and N434A, or            -   T307H and N434H, or            -   T307Q and Q311H, or            -   T307Q and E430H, or            -   T307Q and N434H, or            -   T307Q and N434A, or            -   M252Y and S254T and T256E, if the first Fc-region                polypeptide does not comprises solely the combination                M252Y and S254T and T256E of mutations, or            -   I253A and H310A and H435A, if the first Fc-region                polypeptide does not comprises solely the combination                I253A and H310A and H435A of mutations, or            -   H310A and H433A and Y436A, if the first Fc-region                polypeptide does not comprises solely the combination                H310A and H433A and Y436A of mutations, or            -   T307H and Q311H and E430H and N434A, or            -   T307H and Q311H and E430H and N434H, or            -   T307H and Q311H and E430H and N434Y, or            -   T307Q and Q311H and E430H and N434A, or            -   T307Q and Q311H and E430H and N434H, or            -   T307Q and Q311H and E430H and N434Y, or            -   T307Q and V308P and N434Y and Y436H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations I253A and H310A and H435A and the second Fc-regionpolypeptide comprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations I253A and H310A and H435A and the second Fc-regionpolypeptide comprises the mutations M252Y and S254T and T256E and T307Qand N434Y.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations I253A and H310A and H435A and the second Fc-regionpolypeptide comprises the mutations M252Y and S254T and T256E and T307Qand V308P and N434Y and Y436H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and Q311H and E430H and N434H and the secondFc-region polypeptide comprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307Q and N434A and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T250Q and M428L and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307Q and N434H and the second Fc-region polypeptidecomprises the mutations M252Y and S254T and T256E and T307Q and N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and Q311H and E430H and N434H and the secondFc-region polypeptide comprises the mutations T307H and Q311H and E430Hand N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and the second Fc-region polypeptidecomprises the mutations T307H and N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and M252Y and S254T and T256E and thesecond Fc-region polypeptide comprises the mutations T307H and N434H andM252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutation N434H and the second Fc-region polypeptide comprises themutation N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307Q and N434A.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutations T307H and N434H and M252Y and S254T and T256E.

In one preferred embodiment the first Fc-region polypeptide comprisesthe mutation N434H.

In one embodiment of all aspects the Fc-region is a variant (human) IgGclass Fc-region. In one embodiment the variant (human) IgG classFc-region is an IgG class heterodimeric Fc-region.

In one embodiment of all aspects the pairing of the first Fc-regionpolypeptide and the second Fc-region polypeptide to form a (functional)Fc-region results in the formation of a heterodimer.

In one embodiment the human Fc-region polypeptide is a human Fc-regionpolypeptide of the IgG1 subclass or of the IgG4 subclass.

In one embodiment the human Fc-region polypeptide is a human Fc-regionpolypeptide of the IgG1 subclass which further comprises the mutationsL234A, L235A and P329G.

In one embodiment the human Fc-region polypeptide is a human Fc-regionpolypeptide of the IgG4 subclass which further comprises the mutationsS228P and L235E.

In one embodiment the first Fc-region polypeptide further comprises themutations S354C and T366W and the second Fc-region polypeptide furthercomprises the mutations Y349C, T366S, L368A and Y407V.

In one embodiment the bispecific antibody is characterized in that theFc-region of iii) is of human IgG1 subclass. In one embodiment thebispecific antibody is characterized in that the Fc-region of human IgG1subclass further comprises the mutations L234A, L235A and P329G(numbering according to EU Index of Kabat).

In one embodiment the bispecific antibody is characterized in that theFc-region of iii) is of human IgG4 subclass. In one embodiment thebispecific antibody is characterized in that the Fc-region of human IgG4subclass further comprises the mutations S228P and L235E (numberingaccording to EU Index of Kabat). In one embodiment the bispecificantibody is characterized in that the Fc-region of human IgG4 subclassfurther comprises the mutations S228P, L235E and P329G (numberingaccording to EU Index of Kabat).

Still further aspects as reported herein are a pharmaceuticalformulation comprising the bispecific antibody, the pharmaceuticalformulation for use in the treatment of ocular vascular diseases, theuse of the bispecific antibody for the manufacture of a medicament forthe treatment of ocular vascular diseases, a method of treatment ofpatient suffering from ocular vascular diseases by administering thebispecific antibody to a patient in the need of such treatment. In oneembodiment the bispecific antibody or the pharmaceutical formulationcomprising the bispecific antibody is administered via intravitrealapplication.

A further aspect according to the current invention is a nucleic acidmolecule encoding a heavy and/or light chain of a bispecific antibody asreported herein.

The invention further provides expression vectors containing the nucleicacid as reported herein capable of expressing the nucleic acid in aprokaryotic or eukaryotic host cell, and host cells containing suchvectors for the recombinant production of a bispecific antibody asreported herein.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector as reported herein.

The invention further comprises a method for the production of abispecific antibody as reported herein, characterized by expressing anucleic acid as reported herein in a prokaryotic or eukaryotic host celland recovering the bispecific antibody from the cell or the cell culturesupernatant. One embodiment is a method for the preparation of abispecific antibody as reported herein comprising the steps of

-   -   a) transforming a host cell with vectors comprising nucleic acid        molecules encoding the antibody;    -   b) culturing the host cell under conditions that allow synthesis        of the antibody; and    -   c) recovering the antibody from the culture.

The invention further comprises the antibody obtained by such method forthe production of a bispecific antibody.

The antibodies as reported herein have highly valuable properties due totheir specific modifications in the Fc-region causing a benefit for apatient suffering from ocular vascular diseases. They show highstability in the intravitreal environment and slow diffusion from theeye (compared to smaller antibody fragments without a constant heavychain region), where the actual disease is located and treated (sotreatment schedule may potentially be improved compared to non-IgG likeantibodies like e.g. Fab and (Fab)₂ fragments). The antibodies asreported herein are cleared on the other hand quite rapidly from serum(which is highly desired to reduce potential side effects arising fromsystemic exposure). Surprisingly they also show lower viscosity(compared to versions without the combination of the mutations I253A,H310A and H435A in the constant region) and are therefore especiallyuseful for intravitreal application through thin needles during thetreatment of eye diseases (for such application typically thin needlesare used and high viscosity makes an appropriate application ratherdifficult). The lower viscosity also allows higher concentrationformulations.

Also surprisingly the antibodies as reported herein show a loweraggregation tendency during storage (compared to versions without thecombination of the mutations I253A, H310A and H435A in the Fc-region)which is critical for intravitreal application in the eye (as anaggregation in the eye can lead to complications during such treatment).

The bispecific antibodies as reported herein show good efficacy ininhibition of vascular diseases.

In certain embodiments, the bispecific antibodies as reported herein dueto their specific modifications in the constant region (e.g. P329G LALA)show valuable properties like no binding to/of Fcgamma receptors whichreduces the risk of side effects like thrombosis and/or unwanted celldeath (due to e.g. ADCC).

In one embodiment as reported herein the bispecific antibody as reportedherein is bivalent.

In one embodiment the antibody heavy chain variable domain (VH) and theantibody light chain variable domain (VL) of the heavy and light chainof the second full length antibody are further stabilized by theintroduction of a disulfide bond between the following positions: heavychain variable domain position 44 and light chain variable domainposition 100 (numbering according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).Techniques to introduce disulfide bridges for stabilization aredescribed e.g. in WO 94/029350, Rajagopal, V., et al, Prot. Eng. 10(1997) 1453-1459, Kobayashi et al., Nuclear Medicine & Biology 25 (1998)387-393, and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721.

In one embodiment the CH3 domains of the bispecific, bivalent antibodyas reported herein are altered by the “knob-into-holes” technology whichis described in detail with several examples e.g. in WO 96/027011,Ridgway J. B., et al., Protein Eng. 9 (1996) 617-621, and Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method theinteraction surfaces of the two CH3 domains are altered to increase theheterodimerization of both heavy chains containing these two CH3domains. Each of the two CH3 domains (of the two heavy chains) can bethe “knob-chain” while the other is the “hole-chain”. The introductionof a disulfide bridge further stabilizes the heterodimers (Merchant, A.M, et al., Nature Biotech. 16 (1998) 677-681, Atwell, S., et al. J. Mol.Biol. 270 (1997) 26-35) and increases the yield.

In one preferred embodiment of all aspects as reported herein thebispecific antibodies is characterized in that

-   -   the CH3 domain of one heavy chain and the CH3 domain of the        other heavy chain each meet at an interface which comprises an        original interface between the antibody CH3 domains,    -   wherein the interface is altered to promote the formation of the        bispecific antibody, wherein the alteration is characterized in        that:    -   a) the CH3 domain of one heavy chain is altered,        -   so that within the original interface the CH3 domain of one            heavy chain that meets the original interface of the CH3            domain of the other heavy chain within the bispecific            antibody        -   an amino acid residue is replaced with an amino acid residue            having a larger side chain volume, thereby generating a            protuberance within the interface of the CH3 domain of one            heavy chain which is positionable in a cavity within the            interface of the CH3 domain of the other heavy chain,    -   and    -   b) the CH3 domain of the other heavy chain is altered,        -   so that within the original interface of the second CH3            domain that meets the original interface of the first CH3            domain within the bispecific antibody        -   an amino acid residue is replaced with an amino acid residue            having a smaller side chain volume, thereby generating a            cavity within the interface of the second CH3 domain within            which a protuberance within the interface of the first CH3            domain is positionable.

Thus, the antibody according to invention is in one preferred embodimentcharacterized in that

-   -   the CH3 domain of the heavy chain of the full length antibody        of a) and the CH3 domain of the heavy chain of the full length        antibody of b) each meet at an interface which comprises an        alteration in the original interface between the antibody's CH3        domains,    -   wherein    -   i) in the CH3 domain of one heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a larger side chain volume, thereby generating a        protuberance within the interface of the CH3 domain of one heavy        chain which is positionable in a cavity within the interface of        the CH3 domain of the other heavy chain,    -   and wherein    -   ii) in the CH3 domain of the other heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a smaller side chain volume, thereby generating a cavity        within the interface of the second CH3 domain within which a        protuberance within the interface of the first CH3 domain is        positionable.

In one preferred embodiment the amino acid residue having a larger sidechain volume is selected from the group consisting of arginine (R),phenylalanine (F), tyrosine (Y), tryptophan (W).

In one preferred embodiment the amino acid residue having a smaller sidechain volume is selected from the group consisting of alanine (A),serine (S), threonine (T), valine (V).

In one embodiment both CH3 domains are further altered by theintroduction of a cysteine residue (C) in the corresponding positions ofeach CH3 domain such that a disulfide bridge between both CH3 domainscan be formed.

In one embodiment, the bispecific antibody comprises a T366W mutation inthe CH3 domain of the “knobs chain” and the T366S, L368A and Y407Vmutations in the CH3 domain of the “hole-chain”. An additionalinterchain disulfide bridge between the CH3 domains can also be used(Merchant, A. M, et al., Nature Biotech 16 (1998) 677-681) e.g. byintroducing a Y349C or S354C mutation into the CH3 domain of the “knobschain” and a Y439C or E356C or S354C mutation into the CH3 domain of the“hole chain”.

In one embodiment the bispecific antibody as reported herein comprisesthe mutation Y349C or S354C and the mutation T366W in one of the two CH3domains and the mutations S354C or E356C or Y349C and the mutationsT366S, L368A and Y407V in the other of the two CH3 domains. In onepreferred embodiment the bispecific antibody comprises the Y349C, T366Wmutations in one of the two CH3 domains and the S354C, T366S, L368A,Y407V mutations in the other of the two CH3 domains (the additionalY349C mutation in one CH3 domain and the additional S354C mutation inthe other CH3 domain forming a interchain disulfide bridge) (numberingaccording to EU index of Kabat (Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). In one preferredembodiment the bispecific antibody comprises the S354C, T366W mutationsin one of the two CH3 domains and the Y349C, T366S, L368A, Y407Vmutations in the other of the two CH3 domains (the additional Y349Cmutation in one CH3 domain and the additional S354C mutation in theother CH3 domain forming a interchain disulfide bridge) (numberingaccording to EU index of Kabat (Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)).

But also other knobs-in-holes technologies as described by EP 1 870 459A1, can be used alternatively or additionally. Thus another example forthe bispecific antibody are the R409D and K370E mutations in the CH3domain of the “knobs chain” and the D399K and E357K mutations in the CH3domain of the “hole chain” (numbering according to EU index of Kabat(Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991)).

In another embodiment the bispecific antibody comprises a T366W mutationin the CH3 domain of the “knobs chain” and the T366S, L368A and Y407Vmutations in the CH3 domain of the “hole chain” and additionally theR409D, K370E mutations in the CH3 domain of the “knobs chain” and theD399K, E357K mutations in the CH3 domain of the “hole chain”.

In one embodiment the bispecific antibody comprises the Y349C, T366Wmutations in one of the two CH3 domains and the S354C, T366S, L368A andY407V mutations in the other of the two CH3 domains, or the bispecificantibody comprises the Y349C, T366W mutations in one of the two CH3domains and the S354C, T366S, L368A and Y407V mutations in the other ofthe two CH3 domains and additionally the R409D, K370E mutations in theCH3 domain of the “knobs chain” and the D399K, E357K mutations in theCH3 domain of the “hole chain”.

In one embodiment the bispecific antibody comprises the S354C, T366Wmutations in one of the two CH3 domains and the Y349C, T366S, L368A,Y407V mutations in the other of the two CH3 domains, or the bispecificantibody comprises the S354C, T366W mutations in one of the two CH3domains and the Y349C, T366S, L368A and Y407V mutations in the other ofthe two CH3 domains and additionally the R409D, K370E mutations in theCH3 domain of the “knobs chain” and the D399K, E357K mutations in theCH3 domain of the “hole chain”.

An antigen-binding site of the bispecific antibody as reported hereincontains six complementarity determining regions (CDRs) which contributein varying degrees to the affinity of the binding site for its antigen.There are three heavy chain variable domain CDRs (CDRH1, CDRH2 andCDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 andCDRL3). The extent of CDR and framework regions (FRs) is determined bycomparison to a compiled database of amino acid sequences in which thoseregions have been defined according to variability among the sequences.

In one embodiment of all aspects the antibody does not specifically bindto the human FcRn. In one embodiment of all aspects the antibody inaddition does specifically bind to Staphylococcal protein A.

In one embodiment of all aspects the antibody does not specifically bindto the human FcRn. In one embodiment of all aspects the antibody inaddition does not specifically bind to Staphylococcal protein A

In one embodiment of all aspects the first polypeptide further comprisesthe mutations Y349C, T366S, L368A and Y407V (“hole”) and the secondpolypeptide comprises the mutations S354C and T366W (“knob”).

In one embodiment of all aspects the first polypeptide further comprisesthe mutations S354C, T366S, L368A and Y407V (“hole”) and the secondpolypeptide comprises the mutations Y349C and T366W (“knob”).

In one embodiment of all aspects the Fc-region polypeptides are of thehuman IgG1 subclass. In one embodiment the first Fc-region polypeptideand the second Fc-region polypeptide further comprise the mutationsL234A and L235A. In one embodiment the first Fc-region polypeptide andthe second Fc-region polypeptide further comprise the mutation P329G.

In one embodiment of all aspects the Fc-region polypeptides are of thehuman IgG4 subclass. In one embodiment the first Fc-region polypeptideand the second Fc-region polypeptide further comprise the mutationsS228P and L235E. In one embodiment the first Fc-region polypeptide andthe second Fc-region polypeptide further comprise the mutation P329G.

The antibody as reported herein is produced by recombinant means. Thus,one aspect as reported herein is a nucleic acid encoding the antibody asreported herein and a further aspect is a cell comprising the nucleicacid encoding an antibody as reported herein. Methods for recombinantproduction are widely known in the state of the art and comprise proteinexpression in prokaryotic and eukaryotic cells with subsequent isolationof the antibody and usually purification to a pharmaceuticallyacceptable purity. For the expression of the antibodies asaforementioned in a host cell, nucleic acids encoding the respective(modified) light and heavy chains are inserted into expression vectorsby standard methods. Expression is performed in appropriate prokaryoticor eukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and theantibody is recovered from the cells (cultivation supernatant or cellsafter lysis). General methods for recombinant production of antibodiesare well-known in the state of the art and described, for example, inthe review articles of Makrides, S. C., Protein Expr. Purif. 17 (1999)183-202, Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282,Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-160, and Werner, R. G.,Drug Res. 48 (1998) 870-880.

Accordingly one aspect as reported herein is a method for thepreparation of a bispecific antibody as reported herein, comprising thesteps of

-   -   a) transforming a host cell with vectors comprising nucleic acid        molecules encoding the antibody,    -   b) culturing the host cell under conditions that allow synthesis        of the antibody, and    -   c) recovering the antibody from the culture.

In one embodiment the recovering step under c) includes the use of alight chain constant domain specific capture reagent (which e.g.specific for the kappa or the lambda constant light chain, depending onwhether a kappa or a lambda light chain is contained in the bispecificantibody). In one embodiment this light chain specific capture reagentis used in in a bind-and-elute-mode. Examples of such light chainconstant domain specific capture reagents are e.g. KappaSelect™ andLambdaFabSelect™ (available from GE Healthcare/BAC), which are based ona highly rigid agarose base matrix that allows high flow rates and lowback pressure at large scale. These materials contain a ligand thatbinds to the constant region of the kappa or the lambda light chain,respectively (i.e. fragments lacking the constant region of the lightchain will not bind). Both are therefore capable of binding other targetmolecules containing the constant region of the light chain, forexample, IgG, IgA and IgM. The ligands are attached to the matrix via along hydrophilic spacer arm to make them easily available for binding tothe target molecule. They are based on a single-chain antibody fragmentthat is screened for either human Ig kappa or lambda.

In one embodiment the recovering step under c) includes the use of anFc-region specific capture reagent. In one embodiment the Fc-regionspecific capture reagent is used in a bind-and-elute-mode. Examples ofsuch Fc-region specific capture reagents are e.g. Staphylococcus proteinA-based affinity chromatography materials.

The bispecific antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, affinity chromatography (protein A-Sepharose, or KappaSelect™,LambdaFabSelect™), hydroxylapatite chromatography, gel electrophoresis,or dialysis.

DNA and RNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures. B-cells or hybridoma cells canserve as a source of such DNA and RNA. Once isolated, the DNA may beinserted into expression vectors, which are then transfected into hostcells such as HEK 293 cells, CHO cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofrecombinant monoclonal antibodies in the host cells.

Some of the molecules as reported herein provide ease ofisolation/purification by comprising Fc-regions that are differentiallymodified, wherein at least one of the modifications results in i) adifferential affinity of the molecule for (Staphylococcal) protein A andii) a differential affinity of the molecule for the human FcRn, and themolecule is isolable from a disrupted cell, from medium, or from amixture of molecules based on its affinity for protein A.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art (see e.g. Ausubel, F., et al., ed. CurrentProtocols in Molecular Biology, Greene Publishing and WileyInterscience, New York (1987)). Different methods are well establishedand widespread used for protein purification, such as affinitychromatography with microbial proteins (e.g. protein A or protein Gaffinity chromatography), ion exchange chromatography (e.g. cationexchange (carboxymethyl resins), anion exchange (amino ethyl resins) andmixed-mode exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi,M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

One aspect as reported herein is a pharmaceutical formulation comprisingan antibody as reported herein. Another aspect as reported herein is theuse of an antibody as reported herein for the manufacture of apharmaceutical formulation. A further aspect as reported herein is amethod for the manufacture of a pharmaceutical formulation comprising anantibody as reported herein. In another aspect, a formulation isprovided, e.g. a pharmaceutical formulation, containing an antibody asreported herein, formulated together with a pharmaceutical carrier.

A formulation of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound as reported herein bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

Many possible modes of delivery can be used, including, but not limitedto intraocular application or topical application. In one embodiment theapplication is intraocular and includes, but it's not limited to,subconjunctival injection, intracanieral injection, injection into theanterior chamber via the termporai limbus, intrastromal injection,intracorneal injection, subretinal injection, aqueous humor injection,subtenon injection or sustained delivery device, intravitreal injection(e.g., front, mid or back vitreal injection). In one embodiment theapplication is topical and includes, but it's not limited to eye dropsto the cornea.

In one embodiment the bispecific antibody or pharmaceutical formulationas reported herein is administered via intravitreal application, e.g.via intravitreal injection. This can be performed in accordance withstandard procedures known in the art (see, e.g., Ritter et al., J. Clin.Invest. 116 (2006) 3266-3276, Russelakis-Carneiro et al., Neuropathol.Appl. Neurobiol. 25 (1999) 196-206, and Wray et al., Arch. Neurol. 33(1976) 183-185).

In some embodiments, therapeutic kits as reported herein can contain oneor more doses of a (bispecific) antibody present in a pharmaceuticalformulation described herein, a suitable device for intravitrealinjection of the pharmaceutical formulation, and an instructiondetailing suitable subjects and protocols for carrying out theinjection. In these embodiments, the formulations are typicallyadministered to the subject in need of treatment via intravitrealinjection. This can be performed in accordance with standard proceduresknown in the art (see, e.g., Ritter et al., J. Clin. Invest. 116 (2006)3266-3276, Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25(1999) 196-206, and Wray et al., Arch. Neurol. 33 (1976) 183-185).

The formulations may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the formulations. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds asreported herein, which may be used in a suitable hydrated form, and/orthe pharmaceutical formulations as reported herein, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalformulations as reported herein may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, formulation, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular formulations employed, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular formulations employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The formulation must be sterile and fluid to the extent that theformulation is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the formulation.

The formulation can comprise an ophthalmic depot formulation comprisingan active agent for subconjunctival administration. The ophthalmic depotformulation comprises microparticles of essentially pure active agent,e.g., the bispecific antibody as reported herein. The microparticlescomprising the bispecific antibody as reported herein can be embedded ina biocompatible pharmaceutically acceptable polymer or a lipidencapsulating agent. The depot formulations may be adapted to releaseall of substantially all the active material over an extended period oftime.

The polymer or lipid matrix, if present, may be adapted to degradesufficiently to be transported from the site of administration afterrelease of all or substantially all the active agent. The depotformulation can be liquid formulation, comprising a pharmaceuticalacceptable polymer and a dissolved or dispersed active agent. Uponinjection, the polymer forms a depot at the injections site, e.g. bygelifying or precipitating.

Another aspect as reported herein is the bispecific antibody as reportedherein for use in the treatment of ocular vascular diseases.

Another aspect as reported herein is the pharmaceutical formulation asreported herein for use in the treatment of ocular vascular diseases.

Another aspect as reported herein is the use of an antibody as reportedherein for the manufacture of a medicament for the treatment of ocularvascular disease.

Another aspect as reported herein is method of treatment of patientsuffering from ocular vascular diseases by administering an antibody asreported herein to a patient in the need of such treatment.

It is herewith expressly stated that the term “comprising” as usedherein comprises the term “consisting of”. Thus, all aspects andembodiments that contain the term “comprising” are likewise disclosedwith the term “consisting of”.

Modifications

In a further aspect, an Fc-region or antibody as reported herein as wellas according to any of the above embodiments may incorporate any of thefeatures, singly or in combination, as described in Sections 1-6 below:

1. Antibody Affinity

In one embodiment, Kd is measured using a BIACORE® surface plasmonresonance assay. For example, an assay using a BIACORE®-2000 or aBIACORE®-3000 (GE Healthcare Inc., Piscataway, N.J.) is performed at 25°C. with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CMS, GEHealthcare Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/mL (˜0.2μM) before injection at a flow rate of 5 μL/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block non-reactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μL/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on) (see, e.g.,Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophotometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

2. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA81 (1984) 6851-6855). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I., et al., Nature 332 (1988)323-329; Queen, C., et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V., et al., Methods 36 (2005) 25-34 (describingspecificity determining region (SDR) grafting); Padlan, E. A., Mol.Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F.et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); Osbourn, J.et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83(2000) 252-260 (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J., et al., J. Immunol. 151 (1993)2296-2308; framework regions derived from the consensus sequence ofhuman antibodies of a particular subgroup of light or heavy chainvariable regions (see, e.g., Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Presta, L.G., et al., J. Immunol. 151(1993) 2623-2632); human mature (somatically mutated) framework regionsor human germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

3. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk, M. A. and vande Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg,N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies maybe prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and US 2007/0061900, describing VELOCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described (see, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R., et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P., et al., J. Immunol. 147(1991) 86-95). Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

4. Library-Derived Antibodies

Antibodies as reported herein may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37and further described, e.g., in the McCafferty, J. et al., Nature 348(1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks,J. D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J. D. andBradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol.Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA101 (2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284(2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G., et al., Ann. Rev.Immunol. 12 (1994) 433-455.Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self-antigens without any immunization as described by Griffiths,A. D., et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and US2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

5. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express one or more of the targetantigens. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A., et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A., et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (sFv) dimers (see, e.g. Gruber, Metal., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” comprising an antigen binding site that binds to a first antigenas well as another, different antigen (see, US 2008/0069820, forexample).

The antibody or fragment herein also includes multispecific antibodiesdescribed in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792, and WO 2010/145793.

6. Fc-Region and Antibody Variants

In certain embodiments, amino acid sequence variants of the Fc-regionsor antibodies provided herein are contemplated. For example, it may bedesirable to improve the antigen binding affinity and/or otherbiological properties of the antibody. Amino acid sequence variants ofan Fc-region or antibody may be prepared by introducing appropriatemodifications into the nucleotide sequence encoding the antibody, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of residues within theamino acid sequences of the Fc-region or antibody. Any combination ofdeletion, insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, Fc-region or antibody variants having one ormore amino acid substitutions are provided. Sites of interest forsubstitutional mutagenesis include the HVRs and FRs. Conservativesubstitutions are shown in the Table below under the heading of“preferred substitutions”. More substantial changes are provided in thefollowing Table under the heading of “exemplary substitutions”, and asfurther described below in reference to amino acid side chain classes.Amino acid substitutions may be introduced into an antibody of interestand the products screened for a desired activity, e.g.,retained/improved antigen binding, decreased immunogenicity, or improvedADCC or CDC.

TABLE Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent Fc-region or antibody (e.g. ahumanized or human antibody). Generally, the resulting variant(s)selected for further study will have modifications (e.g., improvements)in certain biological properties (e.g., increased affinity, reducedimmunogenicity) relative to the parent Fc-region or antibody and/or willhave substantially retained certain biological properties of the parentFc-region or antibody. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antibodies displayed on phage and screened for a particularbiological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or residues that contactantigen, with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described, e.g., in Hoogenboom, H. R. etal. in Methods in Molecular Biology 178 (2002) 1-37. In some embodimentsof affinity maturation, diversity is introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, chain shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any antibody variants with the desired affinity. Another methodto introduce diversity involves HVR-directed approaches, in whichseveral HVR residues (e.g., 4-6 residues at a time) are randomized. HVRresidues involved in antigen binding may be specifically identified,e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of anFc-region or antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham, B. C. andWells, J. A., Science 244 (1989) 1081-1085. In this method, a residue orgroup of target residues (e.g., charged residues such as arg, asp, his,lys, and glu) are identified and replaced by a neutral or negativelycharged amino acid (e.g., alanine or polyalanine) to determine whetherthe desired biological property, such as e.g. the interaction of theantibody with antigen, is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an Fc-region or antibody comprisingcomplex to identify contact points can be used. Such contact residuesand neighboring residues may be targeted or eliminated as candidates forsubstitution. Variants may be screened to determine whether they containthe desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean Fc-region or antibody with an N-terminal methionyl residue. Otherinsertional variants of the Fc-region or antibody molecule include thefusion to the N- or C-terminus of the Fc-region or antibody to an enzyme(e.g. for ADEPT) or a polypeptide which increases the serum half-life ofthe Fc-region or antibody.

b) Glycosylation Variants

In certain embodiments, an Fc-region or antibody provided herein isaltered to increase or decrease the extent to which the Fc-region orantibody is glycosylated. Addition or deletion of glycosylation sites toan Fc-region or antibody may be conveniently accomplished by alteringthe amino acid sequence such that one or more glycosylation sites iscreated or removed.

Where the antibody comprises an Fc-region, the carbohydrate attachedthereto may be altered. Native Fc-regions or antibodies produced bymammalian cells typically comprise a branched, biantennaryoligosaccharide that is generally attached by an N-linkage to Asn297 ofthe CH2 domain of the Fc-region. See, e.g., Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32. The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose,and sialic acid, as well as a fucose attached to a GlcNAc in the “stem”of the biantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in an Fc-region or antibody asreported herein may be made in order to create antibody variants withcertain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to anFc-region. For example, the amount of fucose in such antibody may befrom 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc-region (EUnumbering of Fc-region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US 2003/0157108; US 2004/0093621. Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J., et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N., et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y.et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Fc-region or antibody variants are further provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc-region of the antibody is bisected by GlcNAc. Such Fc-regionor antibody variants may have reduced fucosylation and/or improved ADCCfunction. Examples of such antibody variants are described, e.g., in WO2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546. Fc-region orantibody variants with at least one galactose residue in theoligosaccharide attached to the Fc-region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

c) Fc-Region Variants

In certain embodiments, one or more further amino acid modifications maybe introduced into the Fc-region provided herein, thereby generating anFc-region variant. The Fc-region variant may comprise a human Fc-regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc-region) comprisingan amino acid modification (e.g. a substitution/mutation) at one or moreamino acid positions.

In certain embodiments, the invention contemplates an Fc-region variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theFc-region in vivo is important yet certain effector functions (such asCDC and ADCC) are unnecessary or deleterious. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the Fc-region or antibody lacksFcγR binding (hence likely lacking ADCC activity), but retains FcRnbinding ability. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch, J. V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991)457-492. Non-limiting examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83(1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see Bruggemann, M. etal., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactiveassays methods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998)652-656. C1q binding assays may also be carried out to confirm that theFc-region or antibody is unable to bind C1q and hence lacks CDCactivity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro, H. et al., J. Immunol.Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101 (2003)1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103 (2004)2738-2743). FcRn binding and in vivo clearance/half-life determinationscan also be performed using methods known in the art (see, e.g.,Petkova, S. B. et al., Int. Immunol. 18 (2006) 1759-1769).

Fc-regions or antibodies with reduced effector function include thosewith substitution of one or more of Fc-region residues 238, 265, 269,270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc-region variantsinclude Fc-regions with substitutions at two or more of amino acidpositions 265, 269, 270, 297 and 327, including the so-called “DANA”Fc-region mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Certain Fc-region or antibody variants with improved or diminishedbinding to FcRs are described (see, e.g., U.S. Pat. No. 6,737,056; WO2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)6591-6604).

In certain embodiments, an Fc-region or antibody variant comprises anFc-region with one or more amino acid substitutions which improve ADCC,e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region(EU numbering of residues).

In some embodiments, alterations are made in the Fc-region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc-regionwith one or more substitutions therein which improve binding of theFc-region to FcRn. Such Fc-region variants include those withsubstitutions at one or more of Fc-region residues: 238, 256, 265, 272,286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (U.S.Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc-region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc-region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Fc-Region and Antibody Derivatives

In certain embodiments, an Fc-region or antibody provided herein may befurther modified to contain additional non-proteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the Fc-region or antibody include but are not limitedto water soluble polymers. Non-limiting examples of water solublepolymers include, but are not limited to, polyethylene glycol (PEG),copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose,dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or non-branched. The number of polymersattached to the Fc-region or antibody may vary, and if more than onepolymer is attached, they can be the same or different molecules. Ingeneral, the number and/or type of polymers used for derivatization canbe determined based on considerations including, but not limited to, theparticular properties or functions of the Fc-region or antibody to beimproved, whether the Fc-region or antibody derivative will be used in atherapy under defined conditions, etc.

In another embodiment, conjugates of an Fc-region or antibody and one ormore non-proteinaceous moiety that may be selectively heated by exposureto radiation are provided. In one embodiment, the non-proteinaceousmoiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci.USA 102 (2005) 11600-11605). The radiation may be of any wavelength, andincludes, but is not limited to, wavelengths that do not harm ordinarycells, but which heat the non-proteinaceous moiety to a temperature atwhich cells proximal to the antibody-non-proteinaceous moiety arekilled.

f) Heterodimerization

There exist several approaches for CH3-modifications to enforce theheterodimerization, which are well described e.g. in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO2013157954, WO 2013096291. Typically in all such approaches the firstCH3 domain and the second CH3 domains are both engineered in acomplementary manner so that each CH3 domain (or the heavy chaincomprising it) cannot longer homodimerize with itself but is forced toheterodimerize with the complementary engineered other CH3 domain (sothat the first and second CH3 domain heterodimerize and no homodimersbetween the two first or the two second CH3 domains are formed). Thesedifferent approaches for improved heavy chain heterodimerization arecontemplated as different alternatives in combination with theheavy-light chain modifications (VH and VL exchange/replacement in onebinding arm and the introduction of substitutions of charged amino acidswith opposite charges in the CH1/CL interface) in the multispecificantibodies according to the invention which reduce light chainmispairing an Bence-Jones type side products.

In one preferred embodiment of the invention (in case the multispecificantibody comprises CH3 domains in the heavy chains) the CH3 domains ofsaid multispecific antibody according to the invention can be altered bythe “knob-into-holes” technology which is described in detail withseveral examples in e.g. WO 96/027011, Ridgway, J. B., et al., ProteinEng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16(1998) 677-681; WO 98/050431. In this method the interaction surfaces ofthe two CH3 domains are altered to increase the heterodimerization ofboth heavy chains containing these two CH3 domains. Each of the two CH3domains (of the two heavy chains) can be the “knob”, while the other isthe “hole”. The introduction of a disulfide bridge further stabilizesthe heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998)677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) andincreases the yield.

Thus in one embodiment of the invention said multispecific antibody(comprises a CH3 domain in each heavy chain and) is furthercharacterized in that

-   -   the first CH3 domain of the first heavy chain of the antibody        under a) and the second CH3 domain of the second heavy chain of        the antibody under b) each meet at an interface which comprises        an original interface between the antibody CH3 domains.        -   wherein said interface is altered to promote the formation            of the multispecific antibody, wherein the alteration is            characterized in that:        -   i) the CH3 domain of one heavy chain is altered,        -   so that within the original interface of the CH3 domain of            one heavy chain that meets the original interface of the CH3            domain of the other heavy chain within the multispecific            antibody,        -   an amino acid residue is replaced with an amino acid residue            having a larger side chain volume, thereby generating a            protuberance within the interface of the CH3 domain of one            heavy chain which is positionable in a cavity within the            interface of the CH3 domain of the other heavy chain        -   and        -   ii) the CH3 domain of the other heavy chain is altered,        -   so that within the original interface of the second CH3            domain that meets the original interface of the first CH3            domain within the multispecific antibody        -   an amino acid residue is replaced with an amino acid residue            having a smaller side chain volume, thereby generating a            cavity within the interface of the second CH3 domain within            which a protuberance within the interface of the first CH3            domain is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered bythe introduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In one preferred embodiment, said multispecific antibody comprises anamino acid T366W mutation in the first CH3 domain of the “knob-chain”and amino acid T366S, L368A, Y407V mutations in the second CH3 domain ofthe “hole-chain”. An additional interchain disulfide bridge between theCH3 domains can also be used (Merchant, A. M., et al., Nature Biotech.16 (1998) 677-681) e.g. by introducing an amino acid Y349C mutation intothe CH3 domain of the “hole chain” and an amino acid E356C mutation oran amino acid S354C mutation into the CH3 domain of the “knobs chain”.

In one preferred embodiment, said multispecific antibody (whichcomprises a CH3 domain in each heavy chain) comprises amino acid S354C,T366W mutations in one of the two CH3 domains and amino acid Y349C,T366S, L368A, Y407V mutations in the other of the two CH3 domains (theadditional amino acid S354C mutation in one CH3 domain and theadditional amino acid Y349C mutation in the other CH3 domain forming aninterchain disulfide bridge) (numbering according to Kabat).

Other techniques for CH3-modifications to enforcing theheterodimerization are contemplated as alternatives of the invention anddescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment the heterodimerization approach described in EP 1 870459A1, can be used alternatively. This approach is based on the by theintroduction of substitutions/mutations of charged amino acids with theopposite charge at specific amino acid positions of the in the CH3/CH3domain interface between both heavy chains. One preferred embodiment forsaid multispecific antibody are amino acid R409D; K370E mutations in thefirst CH3 domain of the (of the multispecific antibody) and amino acidD399K; E357K mutations in the seconds CH3 domain of the multispecificantibody (numbering according to Kabat).

In another embodiment said multispecific antibody comprises a amino acidT366W mutation in the CH3 domain of the “knobs chain” and amino acidT366S, L368A, Y407V mutations in the CH3 domain of the “hole chain” andadditionally amino acid R409D; K370E mutations in the CH3 domain of the“knobs chain” and amino acid D399K; E357K mutations in the CH3 domain ofthe “hole chain”.

In another embodiment said multispecific antibody comprises amino acidS354C, T366W mutations in one of the two CH3 domains and amino acidY349C, T366S, L368A, Y407V mutations in the other of the two CH3 domainsor said multispecific antibody comprises amino acid Y349C, T366Wmutations in one of the two CH3 domains and amino acid S354C, T366S,L368A, Y407V mutations in the other of the two CH3 domains andadditionally amino acid R409D; K370E mutations in the CH3 domain of the“knobs chain” and amino acid D399K; E357K mutations in the CH3 domain ofthe “hole chain”.

In one embodiment the heterodimerization approach described inWO2013/157953 can be used alternatively. In one embodiment a first CH3domain comprises amino acid T366K mutation and a second CH3 domainpolypeptide comprises amino acid L351D mutation. In a further embodimentthe first CH3 domain comprises further amino acid L351K mutation. In afurther embodiment the second CH3 domain comprises further amino acidmutation selected from Y349E, Y349D and L368E (preferably L368E).

In one embodiment the heterodimerization approach described inWO2012/058768 can be used alternatively. In one embodiment a first CH3domain comprises amino acid L351Y, Y407A mutations and a second CH3domain comprises amino acid T366A, K409F mutations. In a furtherembodiment the second CH3 domain comprises a further amino acid mutationat position T411, D399, S400, F405, N390, or K392 e.g. selected from a)T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W,D399Y or D399K, c S400E, S400D, S400R, or S400K F405I, F405M, F405T,F405S, F405V or F405W N390R, N390K or N390D K392V, K392M, K392R, K392L,K392F or K392E. In a further embodiment a first CH3 domain comprisesamino acid L351Y, Y407A mutations and a second CH3 domain comprisesamino acid T366V, K409F mutations. In a further embodiment a first CH3domain comprises amino acid Y407A mutations and a second CH3 domaincomprises amino acid T366A, K409F mutations. In a further embodiment thesecond CH3 domain comprises a further amino acid K392E, T411E, D399R andS400R mutations.

In one embodiment the heterodimerization approach described inWO2011/143545 can be used alternatively e.g. with the amino acidmodification at a position selected from the group consisting of 368 and409.

In one embodiment the heterodimerization approach described inWO2011/090762 which also uses the knobs-into-holes technology describedabove can be used alternatively. In one embodiment a first CH3 domaincomprises amino acid T366W mutations and a second CH3 domain comprisesamino acid Y407A mutations. In one embodiment a first CH3 domaincomprises amino acid T366Y mutations and a second CH3 domain comprisesamino acid Y407T mutations.

In one embodiment the multispecific antibody is of IgG2 isotype and theheterodimerization approach described in WO2010/129304 can be usedalternatively.

In one embodiment the heterodimerization approach described inWO2009/089004 can be used alternatively. In one embodiment a first CH3domain comprises amino acid substitution of K392 or N392 with anegative-charged amino acid (e.g. glutamic acid (E), or aspartic acid(D), preferably K392D or N392D) and a second CH3 domain comprises aminoacid substitution of D399, E356, D356, or E357 with a positive-chargedamino acid (e.g. Lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K and more preferably D399K and E356K. In a furtherembodiment the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negative-charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with anegative-charged amino acid (e.g. glutamic acid (E), or aspartic acid(D)).

In one embodiment the heterodimerization approach described inWO2007/147901 can be used alternatively. In one embodiment a first CH3domain comprises amino acid K253E, D282K, and K322D mutations and asecond CH3 domain comprises amino acid D239K, E240K, and K292Dmutations.

In one embodiment the heterodimerization approach described inWO2007/110205 can be used alternatively.

Recombinant Methods and Formulations

Fc-regions and antibodies may be produced using recombinant methods andformulations, e.g., as described in U.S. Pat. No. 4,816,567. In oneembodiment, isolated nucleic acid(s) encoding an Fc-region or antibodyas described herein is(are) provided. Such nucleic acid may encode anamino acid sequence comprising the VL and/or an amino acid sequencecomprising the VH of the antibody (e.g., the light and/or heavy chainsof the antibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an antibody as reported herein isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an variant Fc-region, nucleic acidencoding the variant Fc-region, e.g., as described above, is isolatedand inserted into one or more vectors for further cloning and/orexpression in a host cell. Such nucleic acid may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding thevariant Fc-region polypeptides or heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli.). After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized”, resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated Fc-region orantibody are also derived from multicellular organisms (invertebratesand vertebrates). Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains have been identified whichmay be used in conjunction with insect cells, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (HEK293 or 293cells as described, e.g., in Graham, F. L., et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P., et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G., et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

Assays

Antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

In one aspect, an antibody as reported herein is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western Blot,etc.

Immunoconjugates

The invention also provides immunoconjugates comprising an antibody asreported herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos.5,635,483, 5,780,588, and 7,498,298); a dolastatin; a calicheamicin orderivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116,5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman, L. M.et al., Cancer Res. 53 (1993) 3336-3342; and Lode, H. N. et al., CancerRes. 58 (1998) 2925-2928); an anthracycline such as daunomycin ordoxorubicin (see Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523;Jeffrey, S. C. et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362;Torgov, M. Y. et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. etal., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, G. M. etal., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H. D. etal., J. Med. Chem. 45 (2002) 4336-4343; and U.S. Pat. No. 6,630,579);methotrexate; vindesine; a taxane such as docetaxel, paclitaxel,larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example TC^(99m) orI¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (alsoknown as magnetic resonance imaging, MRI), such as iodine-123,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta, E. S. et al., Science 238 (1987)1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari, R. V. et al., Cancer Res. 52 (1992)127-131; U.S. Pat. No. 5,208,020) may be used.

The immuno-conjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone) benzoate) which are commerciallyavailable (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,U.S.A).

Methods and Formulations for Diagnostics and Detection

In certain embodiments, any of the antibodies provided herein is usefulfor detecting the presence of its cognate antigen(s) in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue.

In one embodiment, an antibody as reported herein for use in a method ofdiagnosis or detection is provided.

In certain embodiments, labeled antibodies as reported herein areprovided. Labels include, but are not limited to, labels or moietiesthat are detected directly (such as fluorescent, chromophoric,electron-dense, chemiluminescent, and radioactive labels), as well asmoieties, such as enzymes or ligands, that are detected indirectly,e.g., through an enzymatic reaction or molecular interaction. Exemplarylabels include, but are not limited to, the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, luceriferases, e.g., firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Pharmaceutical Formulations

Pharmaceutical formulations of an antibody as described herein areprepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.)(1980)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyl dimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US 2005/0260186 and US2006/0104968. In one aspect, a sHASEGP is combined with one or moreadditional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Therapeutic Methods and Formulations

Any of the antibodies provided herein may be used in therapeuticmethods.

In one aspect, an antibody as reported herein for use as a medicament isprovided.

In certain embodiments, an antibody for use in a method of treatment isprovided. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. An “individual”according to any of the above embodiments is in one preferred embodimenta human.

In a further aspect, the invention provides for the use of an antibodyin the manufacture or preparation of a medicament. An “individual”according to any of the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the antibodies provided herein, e.g., for use in anyof the above therapeutic methods. In one embodiment, a pharmaceuticalformulation comprises any of the antibodies provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical formulation comprises any of the antibodies providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Antibodies as reported herein can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody as reportedherein may be co-administered with at least one additional therapeuticagent.

An antibody as reported herein (and any additional therapeutic agent)can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies as reported herein would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody as reported herein (when used alone or in combination with oneor more other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate as reported hereinin place of or in addition to an antibody as reported herein.

Articles of Manufacture

In another aspect as reported herein, an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described above is provided. The article ofmanufacture comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a formulation which is by itself or combined withanother formulation effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the formulation is an antibody as reported herein. The label orpackage insert indicates that the formulation is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a formulation contained therein, wherein theformulation comprises an antibody as reported herein; and (b) a secondcontainer with a formulation contained therein, wherein the formulationcomprises a further cytotoxic or otherwise therapeutic agent. Thearticle of manufacture in this embodiment as reported herein may furthercomprise a package insert indicating that the formulations can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate as reported herein in place of or in additionto an antibody as reported herein.

III. Specific Embodiments

-   1. An IgG class Fc-region comprising a first variant Fc-region    polypeptide and a second variant Fc-region polypeptide,    -   wherein    -   a) the first variant Fc-region polypeptide is derived from a        first parent IgG class Fc-region polypeptide and the second        variant Fc-region polypeptide is derived from a second parent        IgG class Fc-region polypeptide, whereby the first parent IgG        class Fc-region polypeptide is identical to or different from        the second parent IgG class Fc-region polypeptide, and    -   b) the first variant Fc-region polypeptide differs from the        second variant Fc-region polypeptide in one or more amino acid        residues other than those amino acid residues in which the first        parent IgG class Fc-region polypeptide differs from the second        parent IgG class Fc-region polypeptide, and    -   c) the IgG class Fc-region comprising the first variant        Fc-region polypeptide and the second variant Fc-region        polypeptide has an affinity to a human Fc-receptor that is        different than that of an IgG class Fc-region comprising the        first parent IgG class Fc-region polypeptide of a) and the        second parent IgG class Fc-region polypeptide of a).-   2. The IgG class Fc-region according to embodiment 1, wherein the    human Fc-receptor is the human neonatal Fc receptor (FcRn) or the    human FcgammaIII receptor (FcγRIII).-   3. The IgG class Fc-region according to any one of embodiments 1 to    2, wherein the human Fc-receptor is the human neonatal Fc-receptor.-   4. The IgG class Fc-region according to any one of embodiments 1 to    3, wherein the affinity of the IgG class Fc-region comprising the    first variant Fc-region polypeptide and the second variant Fc-region    polypeptide to a human Fc-receptor is increased or reduced by 10% or    more determined by surface plasmon resonance (SPR) compared to that    of an IgG class Fc-region comprising the first parent IgG class    Fc-region polypeptide of a) and the second parent IgG class    Fc-region polypeptide of a).-   5. The IgG class Fc-region according to any one of embodiments 1 to    4, wherein at least some of those amino acid residues in which the    first parent IgG class Fc-region polypeptide differs from the second    parent IgG class Fc-region polypeptide promote the formation of a    heterodimeric IgG class Fc-region.-   6. The IgG class Fc-region according to any one of embodiments 1 to    5, wherein    -   i) the first parent IgG class Fc-region polypeptide is selected        from the group comprising        -   human IgG1 Fc-region polypeptide,        -   human IgG2 Fc-region polypeptide,        -   human IgG3 Fc-region polypeptide,        -   human IgG4 Fc-region polypeptide,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A,        -   human IgG1 Fc-region polypeptide with the mutations Y349C,            T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations S354C,            T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, Y349C, T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, S354C, T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations P329G,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, P329G,        -   human IgG1 Fc-region polypeptide with the mutations P329G,            Y349C, T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations P329G,            S354C, T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, P329G, Y349C, T366S, L368A, Y407V,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, P329G, S354C, T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, P329G,        -   human IgG4 Fc-region polypeptide with the mutations Y349C,            T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations S354C,            T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, Y349C, T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, S354C, T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations P329G,        -   human IgG4 Fc-region polypeptide with the mutations P329G,            Y349C, T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations P329G,            S354C, T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, P329G, Y349C, T366S, L368A, Y407V,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, P329G, S354C, T366S, L368A, Y407V,        -   human IgG1, IgG2 or IgG4 with the mutation K392D, and        -   human IgG3 with the mutation N392D,    -   and    -   ii) the second parent IgG class Fc-region polypeptide is        selected from the group comprising        -   human IgG1 Fc-region polypeptide,        -   human IgG2 Fc-region polypeptide,        -   human IgG3 Fc-region polypeptide,        -   human IgG4 Fc-region polypeptide,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A,        -   human IgG1 Fc-region polypeptide with the mutations S354C,            T366W,        -   human IgG1 Fc-region polypeptide with the mutations Y349C,            T366W,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, S354C, T366W,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, Y349C, T366W,        -   human IgG1 Fc-region polypeptide with the mutations P329G,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, P329G,        -   human IgG1 Fc-region polypeptide with the mutations P329G,            S354C, T366W,        -   human IgG1 Fc-region polypeptide with the mutations P329G,            Y349C, T366W,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, P329G, S354C, T366W,        -   human IgG1 Fc-region polypeptide with the mutations L234A,            L235A, P329G, Y349C, T366W,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, P329G,        -   human IgG4 Fc-region polypeptide with the mutations S354C,            T366W,        -   human IgG4 Fc-region polypeptide with the mutations Y349C,            T366W,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, S354C, T366W,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, Y349C, T366W,        -   human IgG4 Fc-region polypeptide with the mutations P329G,        -   human IgG4 Fc-region polypeptide with the mutations P329G,            S354C, T366W,        -   human IgG4 Fc-region polypeptide with the mutations P329G,            Y349C, T366W,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, P329G, S354C, T366W,        -   human IgG4 Fc-region polypeptide with the mutations S228P,            L235E, P329G, Y349C, T366W,        -   human IgG1 with the mutations D399K, D356K, and/or E357K,            and        -   human IgG2, IgG3 or IgG4 with the mutations D399K, E356K,            and/or E357K.-   7. The IgG class Fc-region according to any one of embodiments 1 to    6, wherein    -   i) the first parent IgG class Fc-region polypeptide is a human        IgG1 Fc-region polypeptide and the second parent IgG class        Fc-region polypeptide is a human IgG1 Fc-region polypeptide, or    -   ii) the first parent IgG class Fc-region polypeptide is a human        IgG1 Fc-region polypeptide with the mutations L234A, L235A and        the second parent IgG class Fc-region polypeptide is a human        IgG1 Fc-region polypeptide with the mutations L234A, L235A, or    -   iii) the first parent IgG class Fc-region polypeptide is a human        IgG1 Fc-region polypeptide with the mutations L234A, L235A,        P329G and the second parent IgG class Fc-region polypeptide is a        human IgG1 Fc-region polypeptide with the mutations L234A,        L235A, P329G, or    -   iv) the first parent IgG class Fc-region polypeptide is a human        IgG1 Fc-region polypeptide with the mutations L234A, L235A,        S354C, T366W and the second parent IgG class Fc-region        polypeptide is a human IgG1 Fc-region polypeptide with the        mutations L234A, L235A, Y349C, T366S, L368A, Y407V, or    -   v) the first parent IgG class Fc-region polypeptide is a human        IgG1 Fc-region polypeptide with the mutations L234A, L235A,        P329G, S354C, T366W and the second parent IgG class Fc-region        polypeptide is a human IgG1 Fc-region polypeptide with the        mutations L234A, L235A, P329G, Y349C, T366S, L368A, Y407V, or    -   vi) the first parent IgG class Fc-region polypeptide is a human        IgG4 Fc-region polypeptide and the second parent IgG class        Fc-region polypeptide is a human IgG4 Fc-region polypeptide, or    -   vii) the first parent IgG class Fc-region polypeptide is a human        IgG4 Fc-region polypeptide with the mutations S228P, L235E and        the second parent IgG class Fc-region polypeptide is a human        IgG4 Fc-region polypeptide with the mutations S228P, L235E, or    -   viii) the first parent IgG class Fc-region polypeptide is a        human IgG4 Fc-region polypeptide with the mutations S228P,        L235E, P329G and the second parent IgG class Fc-region        polypeptide is a human IgG4 Fc-region polypeptide with the        mutations S228P, L235E, P329G, or    -   ix) the first parent IgG class Fc-region polypeptide is a human        IgG4 Fc-region polypeptide with the mutations S228P, L235E,        S354C, T366W and the second parent IgG class Fc-region        polypeptide is a human IgG4 Fc-region polypeptide with the        mutations S228P, L235E, Y349C, T366S, L368A, Y407V, or    -   x) the first parent IgG class Fc-region polypeptide is a human        IgG4 Fc-region polypeptide with the mutations S228P, L235E,        P329G, S354C, T366W and the second parent IgG class Fc-region        polypeptide is a human IgG4 Fc-region polypeptide with the        mutations S228P, L235E, P329G, Y349C, T366S, L368A, Y407V.-   8. The IgG class Fc-region according to any one of embodiments 1 to    7, wherein the first Fc-region polypeptide or the second Fc-region    polypeptide or both Fc-region polypeptides comprise one of the    following mutations or combination of mutations:    -   T307H, or    -   Q311H, or    -   E430H, or    -   N434H, or    -   T307H and Q311H, or    -   T307H and E430H, or    -   T307H and N434A, or    -   T307H and N434H, or    -   T307Q and Q311H, or    -   T307Q and E430H, or    -   T307Q and N434H, or    -   T307H and Q311H and E430H and N434A, or    -   T307H and Q311H and E430H and N434H, or    -   T307H and Q311H and E430H and N434Y, or    -   T307Q and Q311H and E430H and N434A, or    -   T307Q and Q311H and E430H and N434H, or    -   T307Q and Q311H and E430H and N434Y, or    -   T307Q and V308P and N434Y and Y436H, or    -   T307H and M252Y and S254T and T256E, or    -   T307Q and M252Y and S254T and T256E, or    -   Q311H and M252Y and S254T and T256E, or    -   E430H and M252Y and S254T and T256E, or    -   N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and M252Y and S254T and T256E, or    -   T307H and E430H and M252Y and S254T and T256E, or    -   T307H and N434A and M252Y and S254T and T256E, or    -   T307H and N434H and M252Y and S254T and T256E, or    -   T307Q and Q311H and M252Y and S254T and T256E, or    -   T307Q and E430H and M252Y and S254T and T256E, or    -   T307Q and N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307Q and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and        T256E.-   9. The IgG class Fc-region according to any one of embodiments 1 to    8, wherein the first Fc-region polypeptide comprise independently of    the second Fc-region polypeptide one of the following mutations or    combination of mutations:    -   T307H, or    -   Q311H, or    -   E430H, or    -   N434H, or    -   T307H and Q311H, or    -   T307H and E430H, or    -   T307H and N434A, or    -   T307H and N434H, or    -   T307Q and Q311H, or    -   T307Q and E430H, or    -   T307Q and N434H, or    -   M252Y and S254T and T256E, or    -   I253A and H310A and H435A, or    -   H310A and H433A and Y436A, or    -   T307H and Q311H and E430H and N434A, or    -   T307H and Q311H and E430H and N434H, or    -   T307H and Q311H and E430H and N434Y, or    -   T307Q and Q311H and E430H and N434A, or    -   T307Q and Q311H and E430H and N434H, or    -   T307Q and Q311H and E430H and N434Y, or    -   T307Q and V308P and N434Y and Y436H, or    -   T307H and M252Y and S254T and T256E, or    -   Q311H and M252Y and S254T and T256E, or    -   E430H and M252Y and S254T and T256E, or    -   N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and M252Y and S254T and T256E, or    -   T307H and E430H and M252Y and S254T and T256E, or    -   T307H and N434A and M252Y and S254T and T256E, or    -   T307H and N434H and M252Y and S254T and T256E, or    -   T307Q and Q311H and M252Y and S254T and T256E, or    -   T307Q and E430H and M252Y and S254T and T256E, or    -   T307Q and N434H and M252Y and S254T and T256E, or    -   T307H and Q311H and E430H and N434A and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434H and M252Y and S254T and        T256E, or    -   T307H and Q311H and E430H and N434Y and M252Y and S254T and        T256E, or    -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and        T256E, or    -   T307Q and V308P and N434Y and Y436H and M252Y and S254T and        T256E,    -   and    -   the second Fc-region polypeptide comprise independently of the        first Fc-region polypeptide one of the following mutations or        combination of mutations        -   T307H, or        -   T307Q, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H, or        -   T307H and M252Y and S254T and T256E, or        -   Q311H and M252Y and S254T and T256E, or        -   E430H and M252Y and S254T and T256E, or        -   N434H and M252Y and S254T and T256E.-   10. The IgG class Fc-region according to any one of embodiments 1 to    9, wherein the first Fc-region polypeptide comprises    -   one of the following combinations of mutations:        -   none, or        -   M252Y and S254T and T256E, or        -   I253A and H310A and H435A, or        -   H310A and H433A and Y436A,    -   and    -   one of the following mutations or combination of mutations:        -   none        -   T307H, or        -   T307Q, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307Q and N434A, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H,    -   and the second Fc-region polypeptide comprises    -   one of the following mutations or combination of mutations:        -   none, if the first Fc-region polypeptide comprises at least            one mutation, or        -   T307H, or        -   T307Q, if the first Fc-region polypeptide does not comprises            solely the T307Q mutation, or        -   Q311H, or        -   E430H, or        -   N434H, or        -   T307H and Q311H, or        -   T307H and E430H, or        -   T307H and N434A, or        -   T307H and N434H, or        -   T307Q and Q311H, or        -   T307Q and E430H, or        -   T307Q and N434H, or        -   T307Q and N434A, or        -   M252Y and S254T and T256E, if the first Fc-region            polypeptide does not comprises solely the combination M252Y            and S254T and T256E of mutations, or        -   I253A and H310A and H435A, if the first Fc-region            polypeptide does not comprises solely the combination I253A            and H310A and H435A of mutations, or        -   H310A and H433A and Y436A, if the first Fc-region            polypeptide does not comprises solely the combination H310A            and H433A and Y436A of mutations, or        -   T307H and Q311H and E430H and N434A, or        -   T307H and Q311H and E430H and N434H, or        -   T307H and Q311H and E430H and N434Y, or        -   T307Q and Q311H and E430H and N434A, or        -   T307Q and Q311H and E430H and N434H, or        -   T307Q and Q311H and E430H and N434Y, or        -   T307Q and V308P and N434Y and Y436H.-   11. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    I253A and H310A and H435A and the second Fc-region polypeptide    comprises the mutations M252Y and S254T and T256E.-   12. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    I253A and H310A and H435A and the second Fc-region polypeptide    comprises the mutations M252Y and S254T and T256E and T307Q and    N434Y.-   13. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    I253A and H310A and H435A and the second Fc-region polypeptide    comprises the mutations M252Y and S254T and T256E and T307Q and    V308P and N434Y and Y436H.-   14. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and Q311H and E430H and N434H and the second Fc-region    polypeptide comprises the mutations M252Y and S254T and T256E.-   15. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and N434H and the second Fc-region polypeptide comprises the    mutations M252Y and S254T and T256E.-   16. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307Q and N434A and the second Fc-region polypeptide comprises the    mutations M252Y and S254T and T256E.-   17. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T250Q and M428L and the second Fc-region polypeptide comprises the    mutations M252Y and S254T and T256E.-   18. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307Q and N434H and the second Fc-region polypeptide comprises the    mutations M252Y and S254T and T256E and T307Q and N434H.-   19. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and Q311H and E430H and N434H and the second Fc-region    polypeptide comprises the mutations T307H and Q311H and E430H and    N434H.-   20. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and N434H and the second Fc-region polypeptide comprises the    mutations T307H and N434H.-   21. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and N434H and M252Y and S254T and T256E and the second    Fc-region polypeptide comprises the mutations T307H and N434H and    M252Y and S254T and T256E.-   22. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutation    N434H and the second Fc-region polypeptide comprises the mutation    N434H.-   23. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307Q and N434A.-   24. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and N434H.-   25. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutations    T307H and N434H and M252Y and S254T and T256E.-   26. The IgG class Fc-region according to any one of embodiments 1 to    10, wherein the first Fc-region polypeptide comprises the mutation    N434H.-   27. An antibody comprising the IgG class Fc-region according to any    one of embodiments 1 to 26.-   28. The antibody according to embodiment 27, wherein the antibody is    a monoclonal antibody.-   29. The antibody according to any one of embodiments 27 to 28,    wherein the antibody is a human, humanized, or chimeric antibody.-   30. The antibody according to any one of embodiments 27 to 29,    wherein the antibody is a bispecific antibody.-   31. The antibody according to any one of embodiments 27 to 30,    wherein the antibody is a bivalent antibody.-   32. An Fc-region fusion polypeptide comprising the IgG class    Fc-region according to any one of embodiments 1 to 26.-   33. A pharmaceutical formulation comprising the antibody according    to any one of embodiments 27 to 31 or the Fc-region fusion    polypeptide according to embodiment 32.-   34. The pharmaceutical formulation according to embodiment 33,    wherein the pharmaceutical formulation is for use in the treatment    of ocular vascular diseases.-   35. The antibody according to any one of embodiments 27 to 31 or the    Fc-region fusion polypeptide according to embodiment 32 for use as a    medicament.-   36. The use according to embodiment 35, wherein the use is for the    treatment of ocular vascular diseases.-   37. The use of the antibody according to any one of embodiments 27    to 31 or the Fc-region fusion polypeptide according to embodiment 32    in the manufacture of a medicament.-   38. The use according to embodiment 37, wherein the use is for the    manufacture of a medicament for the treatment of ocular vascular    disease.-   39. The antibody according to any one of embodiments 27 to 31 or the    Fc-region fusion polypeptide according to embodiment 32 for use in    the treatment of ocular vascular disease.-   40. A method of treatment of patient suffering from ocular vascular    diseases by administering the antibody according to any one of    embodiments 27 to 31 or the Fc-region fusion polypeptide according    to embodiment 32 to a patient in the need of such treatment.

IV. Examples

The following are examples of methods and formulations as reportedherein. It is understood that various other embodiments may bepracticed, given the general description provided above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope as reported herein. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Methods

Electrospray Ionization Mass Spectrometry (ESI-MS)

Protein aliquots (50 μg) were deglycosylated by adding 0.5 μLN-Glycanase plus (Roche) and sodium phosphate buffer (0.1 M, pH 7.1) toobtain a final sample volume of 115 μL. The mixture was incubated at 37°C. for 18 h. Afterwards for reduction and denaturing 60 μL 0.5 M TCEP(Pierce) in 4 M guanidine*HCl (Pierce) and 50 μL 8 M guanidine*HCl wereadded. The mixture was incubated at 37° C. for 30 min. Samples weredesalted by size exclusion chromatography (Sepharose G-25, isocratic,40% acetonitrile with 2% formic acid). ESI mass spectra (+ve) wererecorded on a Q-TOF instrument (maXis, Bruker) equipped with a nano ESIsource (TriVersa NanoMate, Advion). MS parameter settings were asfollows: Transfer: Funnel RF, 400 Vpp; ISCID Energy, 0 eV; Multipole RF,400 Vpp; Quadrupole: Ion Energy, 4.0 eV; Low Mass, 600 m/z; Source: DryGas, 8 L/min; Dry Gas Temperature, 160° C.; Collision Cell: CollisionEnergy, 10 eV; Collision RF: 2000 Vpp; Ion Cooler: Ion Cooler RF, 300Vpp; Transfer Time: 120 μs; Pre Puls Storage, 10 μs; scan range m/z 600to 2000. For data evaluation in-house developed software (MassAnalyzer)was used.

FcRn Surface Plasmon Resonance (SPR) Analysis

The binding properties of wild-type antibody and the mutants to FcRnwere analyzed by surface plasmon resonance (SPR) technology using aBIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This system iswell established for the study of molecular interactions. It allows acontinuous real-time monitoring of ligand/analyte bindings and, thus,the determination of kinetic parameters in various assay settings.SPR-technology is based on the measurement of the refractive index closeto the surface of a gold coated biosensor chip. Changes in therefractive index indicate mass changes on the surface caused by theinteraction of immobilized ligand with analyte injected in solution. Ifmolecules bind to an immobilized ligand on the surface the massincreases, in case of dissociation the mass decreases. In the currentassay, the FcRn receptor was immobilized onto a BIAcore CM5-biosensorchip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling to alevel of 400 Response units (RU). The assay was carried out at roomtemperature with PBS, 0.05% Tween-20™ pH 6.0 (GE Healthcare Bioscience)as running and dilution buffer. 200 nM of antibody samples were injectedat a flow rate of 50 μL/min at room temperature. Association time was180 seconds, dissociation phase took 360 seconds. Regeneration of thechip surface was reached by a short injection of HBS-P, pH 8.0.Evaluation of SPR-data was performed by comparison of the biologicalresponse signal height at 180 seconds after injection and at 300 secondsafter injection. The corresponding parameters are the RU max level (180seconds after injection) and late stability (300 seconds after end ofinjection).

Protein A Surface Plasmon Resonance (SPR) Analysis

The assay is based on surface plasmon resonance spectroscopy. Protein Ais immobilized onto the surface of a SPR biosensor. By injecting thesample into the flow cells of the SPR spectrometer it forms a complexwith the immobilized protein A resulting in an increasing mass on thesensor chip surface, and therefore to a higher response (as 1 RU isdefined as 1 pg/mm²). Afterwards the sensor chip is regenerated bydissolving the sample-protein A-complex. The gained responses are thenevaluated for the signal high in response units (RU) and thedissociation behavior.

Around 3,500 response units (RU) of protein A (20 μg/mL) were coupledonto a CMS chip (GE Healthcare) at pH 4.0 by using the amine couplingkit of GE Healthcare.

The sample and system buffer was HBS-P+ (0.01 M HEPES, 0.15 M NaCl,0.005% Surfactant P20 Sterile-filtered, pH 7.4). Flow cell temperaturewas set to 25° C. and sample compartment temperature to 12° C. Thesystem was primed with running buffer. Then, a 5 nM solutions of thesample constructs were injected for 120 seconds with a flow rate of 30μL/min, followed by a 300 seconds dissociation phase. Then the sensorchip surface was regenerated by two 30 seconds long injections ofGlycine-HCl pH 1.5 at a flow rate of 30 μL/min. Each sample was measuredas a triplicate.

The term “with (the) mutation IHH-AAA” as used herein refers to thecombination of the mutations I253A (Ile253Ala), H310A (His310Ala), andH435A (His435Ala) in a constant heavy chain region of IgG1 or IgG4subclass (numbering according to EU Index of Kabat), the term “with(the) mutation HHY-AAA” as used herein refers the combination of themutations H310A (His310Ala), H433A (His433Ala) and Y436A (Tyr436Ala) ina constant heavy chain region of IgG1 or IgG4 subclass (numberingaccording to EU Index of Kabat), the term “with (the) mutation P329GLALA” as used herein refers to the combination of the mutations L234A(Leu234Ala), L235A (Leu235Ala) and P329G (Pro329Gly) in a constant heavychain region of IgG1 subclass (numbering according to EU Index ofKabat), and the term “with (the) mutation SPLE” as used herein refers tothe combination of the mutations S228P (Ser228Pro) and L235E (Leu235Glu)a constant heavy chain region of IgG4 subclass (numbering according toEU Index of Kabat).

General

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Amino acidresidues of antibody chains are numbered and referred to according to EUnumbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969)78-85; Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were ordered according to given specifications atGeneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies expression plasmids fortransient expression (e.g. in HEK293-F cells) based either on a cDNAorganization with or without a CMV-Intron A promoter or on a genomicorganization with a CMV promoter were used.

The transcription unit of the antibody gene was composed of thefollowing elements:

-   -   unique restriction site(s) at the 5′ end,    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   in the case of the cDNA organization the Intron A sequence,    -   a 5′-untranslated region of a human immunoglobulin gene,    -   a nucleic acid encoding an immunoglobulin heavy chain signal        sequence,    -   a nucleic acid encoding the human antibody chain (wild-type or        with domain exchange) either as cDNA or in genomic organization        with the immunoglobulin exon-intron organization,    -   a 3′ non-translated region with a polyadenylation signal        sequence, and    -   unique restriction site(s) at the 3′ end.

Beside the antibody expression cassette the plasmids contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli,    -   a β-lactamase gene which confers ampicillin resistance in E.        coli., and    -   the dihydrofolate reductase gene from Mus musculus as a        selectable marker in eukaryotic cells.

The nucleic acids encoding the antibody chains were generated by PCRand/or gene synthesis and assembled by known recombinant methods andtechniques by connection of the according nucleic acid segments e.g.using unique restriction sites in the respective vectors. The subclonednucleic acid sequences were verified by DNA sequencing. For transienttransfections larger quantities of the plasmids were prepared by plasmidpreparation from transformed E. coli cultures (Nucleobond AX,Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

The bispecific antibodies were expressed by transient co-transfection ofthe respective expression plasmids in in HEK293-F cells growing insuspension as described below.

Example 1

Expression and Purification

Transient Transfections in HEK293-F System

The monospecific and bispecific antibodies were generated by transienttransfection with the respective plasmids (e.g. encoding the heavy andmodified heavy chain, as well as the corresponding light and modifiedlight chain) using the HEK293-F system (Invitrogen) according to themanufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growingin suspension either in a shake flask or in a stirred fermenter inserum-free FreeStyle™ 293 expression medium (Invitrogen) weretransfected with a mix of the respective expression plasmids and293fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning)HEK293-F cells were seeded at a density of 1*10⁶ cells/mL in 600 mL andincubated at 120 rpm, 8% CO₂. The day after the cells were transfectedat a cell density of approx. 1.5*10⁶ cells/mL with approx. 42 mL of amixture of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA(1 μg/mL) encoding the heavy or modified heavy chain, respectively andthe corresponding light chain in an equimolar ratio and B) 20 mlOpti-MEM with 1.2 mL 293 fectin or fectin (2 μL/mL). According to theglucose consumption glucose solution was added during the course of thefermentation. The supernatant containing the secreted antibody washarvested after 5-10 days and antibodies were either directly purifiedfrom the supernatant or the supernatant was frozen and stored.

Purification

Bispecific antibodies were purified from cell culture supernatants byaffinity chromatography using MabSelectSure-Sepharose™ (for non-IHH-AAAmutants) (GE Healthcare, Sweden) or KappaSelect-Agarose (for IHH-AAAmutants) (GE Healthcare, Sweden), hydrophobic interaction chromatographyusing butyl-Sepharose (GE Healthcare, Sweden) and Superdex 200 sizeexclusion (GE Healthcare, Sweden) chromatography.

Briefly, sterile filtered cell culture supernatants were captured on aMabSelectSuRe resin equilibrated (non-IHH-AAA mutations and wild-typeantibodies) with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25mM sodium citrate at pH 3.0. The IHH-AAA mutants were captured on aKappaSelect resin equilibrated with 25 mM Tris, 50 mM NaCl, pH 7.2,washed with equilibration buffer and eluted with 25 mM sodium citrate pH2.9. The eluted antibody fractions were pooled and neutralized with 2 MTris, pH 9.0. The antibody pools were prepared for hydrophobicinteraction chromatography by adding 1.6 M ammonium sulfate solution toa final concentration of 0.8 M ammonium sulfate and the pH adjusted topH 5.0 using acetic acid. After equilibration of the butyl-Sepharoseresin with 35 mM sodium acetate, 0.8 M ammonium sulfate, pH 5.0, theantibodies were applied to the resin, washed with equilibration bufferand eluted with a linear gradient to 35 mM sodium acetate pH 5.0. The(monospecific or bispecific) antibody containing fractions were pooledand further purified by size exclusion chromatography using a Superdex200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mMhistidine, 140 mM NaCl, pH 6.0. The (monospecific or bispecific)antibody containing fractions were pooled, concentrated to the requiredconcentration using Vivaspin ultrafiltration devices (Sartorius StedimBiotech S.A., France) and stored at −80° C.

Purity and antibody integrity were analyzed after each purification stepby CE-SDS using microfluidic Labchip technology (Caliper Life Science,USA). Five μL of protein solution was prepared for CE-SDS analysis usingthe HT Protein Express Reagent Kit according manufacturer's instructionsand analyzed on LabChip GXII system using a HT Protein Express Chip.Data were analyzed using LabChip GX Software.

The aggregate content of antibody samples was analyzed byhigh-performance SEC using a Superdex 200 analytical size-exclusioncolumn (GE Healthcare, Sweden) in 2×PBS (20 mM Na₂HPO₄, 2 mM KH₂PO₄, 274mM NaCl and 5.4 mM KCl, pH 7.4) running buffer at 25° C. 25 μg proteinwere injected on the column at a flow rate of 0.75 mL/min and elutedisocratic over 50 minutes.

Example 2

FcRn Chromatography

Coupling to Streptavidin Sepharose:

One gram streptavidin sepharose (GE Healthcare) was added to thebiotinylated and dialyzed receptor and incubated for two hours withshaking. The receptor derivatized sepharose was filled in a 1 mL XKcolumn (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

Conditions:

-   column dimensions: 50 mm×5 mm-   bed height: 5 cm-   loading: 50 μg sample-   equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH    5.5-   elution buffer: 20 mM Tris/HCl, with 150 mM NaCl, adjusted to pH 8.8-   elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution    buffer, 10 CV elution buffer

The invention claimed is:
 1. An IgG class Fc-region comprising a firstvariant Fc-region polypeptide and a second variant Fc-regionpolypeptide, wherein: a) the first variant Fc-region polypeptide isderived from a first parent IgG class Fc-region polypeptide and thesecond variant Fc-region polypeptide is derived from a second parent IgGclass Fc-region polypeptide, whereby the first parent IgG classFc-region polypeptide is identical to or different from the secondparent IgG class Fc-region polypeptide, and b) the first variantFc-region polypeptide differs from the second variant Fc-regionpolypeptide in one or more amino acid residues other than those aminoacid residues in which the first parent IgG class Fc-region polypeptidediffers from the second parent IgG class Fc-region polypeptide, and c)the IgG class Fc-region comprising the first variant Fc-regionpolypeptide and the second variant Fc-region polypeptide has an affinityto a human neonatal Fc-receptor that is different than that of an IgGclass Fc-region comprising the first parent IgG class Fc-regionpolypeptide of a) and the second parent IgG class Fc-region polypeptideof a), wherein either the first variant Fc-region polypeptide or thesecond variant Fc-region polypeptide or both variant Fc-regionpolypeptides comprise independently of each other T307Q and V308P andN434Y and Y436H.
 2. The IgG class Fc-region according to claim 1,wherein the amino acid residues in which the first parent IgG classFc-region polypeptide differs from the second parent IgG class Fc-regionpolypeptide promote the formation of a heterodimeric IgG classFc-region.
 3. The IgG class Fc-region according to claim 1, wherein: i)the first parent IgG class Fc-region polypeptide is selected from thegroup consisting of: a human IgG1 Fc-region polypeptide, a human IgG2Fc-region polypeptide, a human IgG3 Fc-region polypeptide, a human IgG4Fc-region polypeptide, a human IgG1 Fc-region polypeptide comprising themutations L234A and L235A, a human IgG1 Fc-region polypeptide comprisingthe mutations Y349C, T366S, L368A, and Y407V, a human IgG1 Fc-regionpolypeptide comprising the mutations L234A, L235A, Y349C, T366S, L368A,and Y407V, a human IgG1 Fc-region polypeptide comprising the mutationP329G, a human IgG1 Fc-region polypeptide comprising the mutationsL234A, L235A, and P329G, a human IgG1 Fc-region polypeptide comprisingthe mutations P329G, Y349C, T366S, L368A, and Y407V, a human IgG1Fc-region polypeptide comprising the mutations L234A, L235A, P329G,Y349C, T366S, L368A, and Y407V, a human IgG4 Fc-region polypeptidecomprising the mutations S228P and L235E, a human IgG4 Fc-regionpolypeptide comprising the mutations S228P, L235E, and P329G, a humanIgG4 Fc-region polypeptide comprising the mutations Y349C, T366S, L368A,and Y407V, a human IgG4 Fc-region polypeptide comprising the mutationsS228P, L235E, Y349C, T366S, L368A, and Y407V, a human IgG4 Fc-regionpolypeptide comprising the mutation P329G, a human IgG4 Fc-regionpolypeptide comprising the mutations P329G, Y349C, T366S, L368A, andY407V, a human IgG4 Fc-region polypeptide comprising the mutationsS228P, L235E, P329G, Y349C, T366S, L368A, and Y407V, a human IgG1, IgG2or IgG4 comprising the mutation K392D, and a human IgG3 comprising themutation N392D (Kabat EU index numbering system); and ii) the secondparent IgG class Fc-region polypeptide is selected from the groupconsisting of: a human IgG1 Fc-region polypeptide, a human IgG2Fc-region polypeptide, a human IgG3 Fc-region polypeptide, a human IgG4Fc-region polypeptide, a human IgG1 Fc-region polypeptide comprising themutations L234A and L235A, a human IgG1 Fc-region polypeptide comprisingthe mutations S354C and T366W, a human IgG1 Fc-region polypeptidecomprising the mutations L234A, L235A, S354C, and T366W, a human IgG1Fc-region polypeptide comprising the mutation P329G, a human IgG1Fc-region polypeptide comprising the mutations L234A, L235A, and P329G,a human IgG1 Fc-region polypeptide comprising the mutations P329G,S354C, and T366W, a human IgG1 Fc-region polypeptide comprising themutations L234A, L235A, P329G, S354C, and T366W, a human IgG4 Fc-regionpolypeptide comprising the mutations S228P and L235E, a human IgG4Fc-region polypeptide comprising the mutations S228P, L235E, and P329G,a human IgG4 Fc-region polypeptide comprising the mutations S354C andT366W, a human IgG4 Fc-region polypeptide comprising the mutationsS228P, L235E, S354C, and T366W, a human IgG4 Fc-region polypeptidecomprising the mutation P329G, a human IgG4 Fc-region polypeptidecomprising the mutations P329G, S354C, and T366W, a human IgG4 Fc-regionpolypeptide comprising the mutations S228P, L235E, P329G, S354C, andT366W, a human IgG1 comprising one or more of the mutations D399K,D356K, and E357K, and a human IgG2, IgG3 or IgG4 comprising one or moreof the mutations D399K, E356K, and E357K (Kabat EU index numberingsystem).
 4. The IgG class Fc-region according to claim 1, wherein: i)the first parent IgG class Fc-region polypeptide is a human IgG1Fc-region polypeptide and the second parent IgG class Fc-regionpolypeptide is a human IgG1 Fc-region polypeptide; or ii) the firstparent IgG class Fc-region polypeptide is a human IgG1 Fc-regionpolypeptide comprising the mutations L234A and L235A, and the secondparent IgG class Fc-region polypeptide is a human IgG1 Fc-regionpolypeptide comprising the mutations L234A and L235A (Kabat EU indexnumbering system); or iii) the first parent IgG class Fc-regionpolypeptide is a human IgG1 Fc-region polypeptide comprising themutations L234A, L235A, and P329G, and the second parent IgG classFc-region polypeptide is a human IgG1 Fc-region polypeptide comprisingthe mutations L234A, L235A, and P329G (Kabat EU index numbering system);or iv) the first parent IgG class Fc-region polypeptide is a human IgG1Fc-region polypeptide comprising the mutations L234A, L235A, S354C, andT366W, and the second parent IgG class Fc-region polypeptide is a humanIgG1 Fc-region polypeptide comprising the mutations L234A, L235A, Y349C,T366S, L368A, and Y407V (Kabat EU index numbering system); or v) thefirst parent IgG class Fc-region polypeptide is a human IgG1 Fc-regionpolypeptide comprising the mutations L234A, L235A, P329G, S354C, andT366W, and the second parent IgG class Fc-region polypeptide is a humanIgG1 Fc-region polypeptide comprising the mutations L234A, L235A, P329G,Y349C, T366S, L368A, and Y407V (Kabat EU index numbering system); or vi)the first parent IgG class Fc-region polypeptide is a human IgG4Fc-region polypeptide and the second parent IgG class Fc-regionpolypeptide is a human IgG4 Fc-region polypeptide; or vii) the firstparent IgG class Fc-region polypeptide is a human IgG4 Fc-regionpolypeptide comprising the mutations S228P and L235E, and the secondparent IgG class Fc-region polypeptide is a human IgG4 Fc-regionpolypeptide comprising the mutations S228P and L235E (Kabat EU indexnumbering system); or viii) the first parent IgG class Fc-regionpolypeptide is a human IgG4 Fc-region polypeptide comprising themutations S228P, L235E, and P329G, and the second parent IgG classFc-region polypeptide is a human IgG4 Fc-region polypeptide comprisingthe mutations S228P, L235E, and P329G (Kabat EU index numbering system);or ix) the first parent IgG class Fc-region polypeptide is a human IgG4Fc-region polypeptide comprising the mutations S228P, L235E, S354C, andT366W, and the second parent IgG class Fc-region polypeptide is a humanIgG4 Fc-region polypeptide comprising the mutations S228P, L235E, Y349C,T366S, L368A, and Y407V (Kabat EU index numbering system); or x) thefirst parent IgG class Fc-region polypeptide is a human IgG4 Fc-regionpolypeptide comprising the mutations S228P, L235E, P329G, S354C, andT366W, and the second parent IgG class Fc-region polypeptide is a humanIgG4 Fc-region polypeptide comprising the mutations S228P, L235E, P329G,Y349C, T366S, L368A, and Y407V (Kabat EU index numbering system); or xi)the first parent IgG class Fc-region polypeptide is: (a) a human IgG1,IgG2, or IgG4 Fc-region polypeptide comprising the mutation K392D, or(b) a human IgG3 Fc-region polypeptide comprising the mutation N392D,and the second parent IgG class Fc-region polypeptide is: (c) a humanIgG1 Fc-region polypeptide comprising one or more of the mutationsD399K, D356K, and E357K or (d) a human IgG2, IgG3 or IgG4 Fc-regionpolypeptide comprising one or more of the mutations D399K, E356K, andE357K (Kabat EU index numbering system).
 5. An antibody comprising theIgG class Fc-region according to claim
 1. 6. The antibody according toclaim 5, wherein the antibody is a monoclonal antibody.
 7. The antibodyaccording to claim 5, wherein the antibody is a human, humanized, orchimeric antibody.
 8. The antibody according to claim 5, wherein theantibody is a bispecific antibody.
 9. The antibody according to claim 5,wherein the antibody is a bivalent antibody.
 10. A pharmaceuticalformulation comprising the antibody according to claim
 5. 11. A methodfor making an IgG class Fc-region, the method comprising: a) cultivatinga mammalian cell comprising one or more nucleic acids encoding the firstand second variant Fc-region polypeptides according to claim 1; and b)recovering the IgG class Fc-region from the cultivation medium.
 12. Themethod of claim 11, wherein the mammalian cell is a CHO cell.